Journal of Applied Physiology Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 82: 1939-1945, 1997;
8750-7587/97 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cerniglia, G. J.
Right arrow Articles by Biaglow, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cerniglia, G. J.
Right arrow Articles by Biaglow, J.

Journal of Applied Physiology
Vol. 82, No. 6, pp. 1939-1945, June 1997
METABOLISM

Intravascular oxygen distribution in subcutaneous 9L tumors and radiation sensitivity

George J. Cerniglia1, David F. Wilson2, Marek Pawlowski3, Sergei Vinogradov2, and John Biaglow1

Departments of 1 Radiation Oncology and of 2 Biochemistry and Biophysics, Medical School, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and 3 Medical Systems Corporation, Greenvale, New York 11548

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES

ABSTRACT

Cerniglia, George J., David F. Wilson, Marek Pawlowski, Sergei Vinogradov, and John Biaglow. Intravascular oxygen distribution in subcutaneous 9L tumors and radiation sensitivity. J. Appl. Physiol. 82(6): 1939-1945, 1997.---Phosphorescence quenching was evaluated as a technique for measuring PO2 in tumors and for determining the effect of increased PO2 on sensitivity of the tumors to radiation. Suspensions of cultured 9L cells or small pieces of solid tumors from 9L cells were injected subcutaneously on the hindquarter of rats, and tumors were grown to between 0.2 and 1.0 cm in diameter. Oxygen-dependent quenching of the phosphorescence of intravenously injected Pd-meso-tetra-(4-carboxyphenyl) porphine was used to image the in vivo distribution of PO2 in the vasculature of small tumors and surrounding tissue. Maps (512 × 480 pixels) of tissue oxygen distribution showed that the PO2 within 9L tumors was low (2-12 Torr) relative to the surrounding muscle tissue (20-40 Torr). When the rats were given 100% oxygen or carbogen (95% O2-5% CO2) to breathe, the PO2 in the tumors increased significantly. This increase was variable among tumors and was greater with carbogen compared with 100% oxygen. Based on irradiation and regrowth studies, carbogen breathing increased the sensitivity of the tumors to radiation. This is consistent with the measured increase in PO2 in the tumor vasculature. It is concluded that phosphorescence quenching can be used for noninvasive determination of the oxygenation of tumors. This method for oxygen measurements has great potential for clinical application in tumor identification and therapy.

phosphorescence; imaging; glioma

INTRODUCTION

THE PO2 in tumors is an important determinant of their response to radiation and other therapeutic treatments. There is high intratumor heterogeneity in the PO2 as well as variability between tumor types and sizes, making it difficult to extrapolate data from one tumor to another. Thus it is important to be able to determine oxygen values in each individual tumor and to relate these values to the efficacy of the therapy.

There are currently several methods being used for measuring oxygen in experimental tumors, including polarographic needle electrodes (15), phosphorescence quenching (29, 33), and nitroaromatic binding (4-6, 16-18). Of the available methods, only phosphorescence quenching and nitroaromatic binding accurately respond to PO2 values throughout the concentration range that affects radiation therapy. Oxygen electrodes, the most commonly used method for measuring oxygen, do not perform well at low PO2 (<10 Torr) and damage the tissue as they are inserted (e.g., Refs. 15, 23, 27). A "hypoxic fraction" has been defined for tumors on the basis of their radiation sensitivity (for a review and critical analysis of the method, see Ref. 22). The relationship of this calculated hypoxic fraction to tumor PO2 is, however, not well established. The hypoxic fraction of solid 9L tumors has been reported to be low (<3%) and not statistically different from zero (19, 30, 32). This has been interpreted as indicating that the PO2 in solid 9L tumors does not fall below the level required for maximal oxygen sensitivity to radiation, ~5 Torr. On the other hand, measurements of PO2 have indicated there are regions of substantial hypoxia (4, 33).

Oxygen-dependent quenching of phosphorescence provides a noninvasive measurement of vascular PO2 in tissue (22, 33, 37, 38). Used with a video-imaging system, it can provide, in real time, quantitative, two-dimensional maps of the PO2 in the blood contained in the surface tissue to a depth of 0.5-1 mm. These maps can be generated with a resolution of <20 µm/pixel and thus show heterogeneity in oxygen distribution within tissue. In an earlier paper (33), it was reported that PO2 maps of tissue regions containing subcutaneous 9L tumors revealed that the PO2 values in the tumor vasculature were often in the range 2-8 Torr, much below those of 20-40 Torr in the surrounding tissue. In the present study, the response of the oxygen distribution in subcutaneous 9L tumors to altering the inspired gas to 100% oxygen or 95% O2-5% CO2 (carbogen) has been evaluated. We have also demonstrated that breathing carbogen during irradiation increases the radiation induced delay in regrowth.

MATERIALS AND METHODS

Tumor growth in rats. All animal procedures were in accordance with the "Guide for the Care and Use of Laboratory Animals" [DHEW Publication No. (NIH) 86-21, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20892] and were approved by the local Animal Care Committee. The donor tumors used in the present study were initiated by subcutaneous inoculation of ~1 × 106 9L glioma cells in the hindquarter of a male Fischer rat. When the donor tumor had grown to ~1-1.5 cm in diameter, the rat was killed. The tumor was removed and sliced into <2 × 2 × 2-mm pieces. The tumor pieces were then used to initiate new tumors by trocar injection of a piece into a subcutaneous site over the thigh muscle of other rats. These tumors were allowed to grow from 8 to 12 days until they were between 0.5 and 1.2 cm in diameter and 3-5 mm in thickness.

Preparation of animals for oxygen imaging. On the day of the experiment, the animals were anesthetized by using ketamine (80 mg/kg), xylazine (5 mg/kg), and atropine (0.01 mg/kg) by intraperitoneal injection. The animals were placed on a heated pad, and their temperature was maintained at 37-38°C. Approximately 4 mg of Pd-meso-tetra-(4-carboxyphenyl) porphine (Porphyrin Products, Logan, UT) was injected into the tail vein as a solution of 8.5 mg/ml in saline with 60 mg/ml bovine serum albumin (fraction V, ICN ImmunoBiologicals, Costa Mesa, CA), pH 7.4. The skin was dissected away from the tumor surface and a surrounding area of ~2.5 cm in diameter (minimal bleeding was noticed). The tissues were kept moist by bathing the surface with a small amount of saline solution and covering it with a clear plastic film. This plastic film did not interfere with observations of phosphorescence and was left in place during the measurements. Measurements of PO2 in regions of tissue ~1 cm from the edge of tumors were used as the values for normal tissue.

Preparation of animals for tumor irradiation. On the day of the irradiation, the tumors (6/treatment group) were measured with calipers to determine their volume before treatment. The rats were anesthetized as described above and shielded with lead so that only the tumor area was irradiated. Body temperature was maintained by placing the animals on a Deltaphane isothermal pad heated to 37°C. During irradiation, the rats were placed in a modular incubator chamber (Billups-Rothenbrer, Del Mar, CA) and either allowed to breathe either air or carbogen for 10 min before and while receiving 0, 10, or 20 gray (Gy; 2 Gy/min, 0.35-mm Cu filter, 225 KeV). After irradiation, the animals were returned to the colony, and their tumor growth was assessed by caliper measurements each 2 days until they had either grown to 200 mm3 or 22 days. The tumor volumes were calculated as V = a · b · cpi /6, where a and b are the longest and shortest perpendicular diameters, and c is the height.

Measurements of oxygen distribution in tumors. Oxygen was measured by the oxygen-dependent quenching of phosphorescence.

The oxygen dependence of the phosphorescence of the probes can be quantitatively described by the Stern-Volmer relationship
<IT>T</IT><SUP>0</SUP>/<IT>T</IT> = 1 + <IT>k</IT><SUB>Q</SUB><IT>T</IT><SUP>0</SUP>P<SC>o</SC><SUB>2</SUB> (1)
For the Pd complex of tetra-(4-carboxyphenyl) porphine bound to bovine serum albumin, at 36°C and pH values from 6.8 to 7.4, kQ is a second-order rate constant with a value of 350 Torr/s, and T0 is 650 µs (see Ref. 20). T0 and T are the phosphorescence lifetimes at zero PO2 and at a given PO2, respectively. Oxygen is the only compound in normal blood that significantly quenches the phosphorescence of the oxygen probe. Calibration is "absolute" in the sense that for the probe bound to albumin and at a given pH and temperature, the values of kQ and T0 are characteristic of that probe. In our laboratory, injection of the probe has not caused alterations in the blood pressure, arterial PO2 and PCO2, blood glucose, or electroencephalogram in adult cats, rats, mice, or newborn piglets. There have been unpublished reports, by others, of adverse reactions on injection of the phosphor solution, probably to the bovine serum albumin, in adult pigs. The optical filters for measuring phosphorescence (excitation through an interference filter with a center wavelength of 524 nm and a bandwidth at half-height of 40 nm; emission through a 645-nm-long pass filter) were put in place, and the room was darkened. Under these conditions, there was no detectable phosphorescence before the Pd-meso-tetra-(4-carboxyphenyl) porphine was injected.

The illuminating light for the epifluorescence attachment was an EG&G xenon flashlamp (Salem, MA), with a flash duration of <5 µs, mounted in a Leitz lamp housing. The flashlamp was controlled by an 80486 microcomputer (Spear Technology, Northbrook, IL) that determined the timing of the flashes and gating of the video camera intensifier by using a five-channel counter-timer board. The frame-grabber and image-processing software were either a customized version of the Image 1/AT system (Universal Imaging, Malvern, PA) or, more recently, an imaging system developed by Pawlowski and Wilson (Medical Systems, Greenvale, NY). A typical image-collection protocol was as follows: number of frames averaged for each delay time, 8; delay times after the flash, 20, 40, 80, 160, 300, 600, and 2,500 µs; gate width in all cases, 2,500 µs. The image processor averaged the frames for each delay time, and this image was displayed and recorded. Between 1 and 1.5 s were required for the image for each delay time, and ~20 s were required for a complete set of seven images. A data-analysis software system (Vinogradov and Wilson, unpublished observations) was used to calculate the phosphorescence lifetime for each pixel location of the image sequence by best fit to a single exponential. The calculated pixel maps were initially calculated in floating point and then converted to 16-bit images. The data histograms were calculated for the 16-bit images, and then the images were reduced to 8 bits for display and storage. Pixel maps were calculated for the initial phosphorescence intensity (intensity at time 0), phosphorescence lifetime, goodness of fit to a single exponential (correlation coefficient), and PO2. For most of the area of the maps, there is an excellent fit to a single exponential. The frame grabber digitized in only 8 bits (256 gray levels), and in less-bright regions of the phosphorescence images the initial intensities may be below the ~20-gray levels necessary for accurate determination of the decay constant.

The routine that calculates fit to a single exponential also calculates the phosphorescence intensity at time 0 (initial value) for each pixel location. These maps make it possible to relate the measured phosphorescence lifetimes and PO2 to physiological structures observable in images of phosphorescence intensity. The intensity of phosphorescence emitted from the tissue is dependent on the concentration of phosphor in the observed tissue area (proportional to blood volume), the intensity of the excitation light, and the absorbance of the tissue for the excitation light. Phosphorescence measurements require excitation of the phosphor, and the green light used in this study penetrates 0.5-1 mm into the tissue. The emitted light is in the near infrared (maximum at 695 nm), and light of this wavelength is much less absorbed by the tissue. The decrease in phosphorescence intensity toward the edges of the images is due, in part, to lower intensity of illumination by the excitation light at the edge of the illuminated area and partly to tissue curvature. The macroscope was focused on the tissue in the central part of the observed area, and the edges were sometimes below the focal plane.
Fig. 1. A-F: PO2 maps of subcutaneous 9L tumors determined by phosphorescence lifetime. Phosphorescence was imaged according to time delay sequence given in MATERIALS AND METHODS. Images at different delay times were used to calculate fit to a single exponential decay for each pixel region of imaged area. These phosphorescence lifetime maps were then converted to PO2 maps by using Eq. 1. A, C, E, and F: tumor regions from 3 different rats measured while they were breathing air. B: same rat as in A but taken 5 min after changing to 100% O2. D: same rat as in C but taken 5 min after changing to 95% O2-5% CO2 (carbogen) for inspired gasses, respectively. E and F: rat breathing air and with PO2 maps determined ~5 min apart. Maps are for a region 1 cm wide and 0.8 cm high. They were calculated in 256 gray levels with scale for conversion to PO2 shown on right of each O2 map.
[View Larger Version of this Image (93K GIF file)]

RESULTS

Imaging of phosphorescence and measurement of PO2. Phosphorescence was imaged as described above. The phosphorescence from tumor tissue was generally greater than that from the surrounding muscle, a difference that increased as the delay time after the flash increased. Muscle does not have well-defined vessels or other features easily identified in the images of phosphorescence intensity. In some experiments, therefore, black threads were laid across the surface of the tissue to allow the microscope to be focused on the tissue surface as well as for positioning of features in the phosphorescence lifetime and oxygen maps in relation to the tumor. The decrease in phosphorescence with increasing delay after the flash was slower in regions of tumor tissue than in the surrounding tissue, indicating increased phosphorescence lifetimes. Maps of PO2 in tumor regions ~0.8 cm in diameter from two different rats are shown in Fig. 1, A-D. The maps for rats breathing air (A and C) show that the PO2 values in the tumors were lower than control tissue (as was previously reported) (33). When the rats were given 100% oxygen (Fig. 1B) or carbogen (Fig. 1D) to breathe for 10 and 12 min, respectively, there was an increase in the PO2 in both the tumor and normal tissue regions. The central tumor portion had PO2 less than those in the periphery. When the rats were breathing oxygen or carbogen, PO2 increase appeared to be greater in the tumor periphery than near the core.

There were usually regions of what appeared to be normal tissue surrounding the tumor in which the PO2 values were substantially lower than those in tissue further from the tumor. Oxygen maps taken over a period of a few minutes often show fluctuations in pressure in these regions adjacent to tumors. The PO2 maps in Fig. 1, E and F, are of the same region taken ~5 min apart. The primary tumor area in the lower central portion of the map is ~2.5 mm in diameter, and a region of below normal PO2 extends well away from the primary tumor. Many tumors had such adjacent regions of hypoxic tissue, and these hypoxic regions can have significant alterations in PO2 over time. The fluctuations in PO2 appear to be random, and at later times the PO2 has again increased.

Quantitation of distributions of PO2 in tumors in 100% oxygen- or carbogen-breathing rats. The distribution of PO2 within any area of the map can be represented as a histogram, with the number of pixels having each PO2 plotted against PO2 (Fig. 2A). Such histograms accurately present the distribution of PO2 in the selected tissue area. Another presentation format is the integral of the histogram (see Fig. 2B), which can conveniently be expressed as the percentage of the pixels with a PO2 less than the indicated value. The values tissue area contained primarily tumor tissue. The PO2 values in the tumor are heterogeneous but all below the 30-40 Torr characteristic of normal tissue. The rat was given 100% oxygen to breathe for 1, 3, and 5 min, and the responses are representative of those for oxygen breathing as long as 15 min. At longer times the tissue PO2 tended to return toward control values. On the rats being returned to breathing air, the PO2 in the tumor returned to control values. Sometimes there was a temporary undershoot, to values below control, followed by increase again to control values, a process that was usually complete in ~15 min. In a series of different rats, the PO2 in the tumors always increased in response to oxygen breathing, although the rate and extent varied among animals.

Fig. 2. A and B: alterations in O2 distributions in a subcutaneous 9L tumor in response to rat breathing pure O2. PO2 maps such as those shown in Fig. 1 were obtained and tumor region transformed into a histogram (A) by summing number of pixels having each level of PO2 (16-bit image) and plotting this value (ordinate) against PO2 (abscissa) (B) and then integrating histogram to give %pixels with PO2 values less than PO2 value on ordinate. Three curves are PO2 maps with rat breathing air and 1, 3, and 5 min after animal was given 100% O2 to breathe.
[View Larger Versions of these Images (19 + 19K GIF file)]

Rats given carbogen to breathe for periods of 3, 7, and 10 min (Fig. 3, A and B) showed responses similar to those with 100% oxygen. In general, the increase in PO2 in both normal and tumor tissue was greater with carbogen breathing than with 100% oxygen.

Fig. 3. A and B: effect of breathing carbogen on distribution of PO2 values in subcutaneous 9L tumors. PO2 maps of tumors were measured while rat was breathing air (air-1). Rat was then given carbogen to breathe by flowing gas into a cone loosely covering nose. PO2 maps were measured at 3, 7, and 10 min after flow of carbogen gas was started. Flowing gas was then changed back to air, and after 10 min another PO2 map was measured (air-2). Each PO2 map was converted to a histogram (A) and then integrated to give %pixels with PO2 values less than value on ordinate (B).
[View Larger Versions of these Images (22 + 21K GIF file)]

Radiation sensitivity of tumors in animals breathing carbogen. The ability to substantially increase PO2 in the tumor by giving the rats carbogen to breath offered the opportunity to determine whether the observed increase in PO2 would affect the sensitivity of the tumor to radiation. The growth curves for 9L tumors under control conditions, as well as after 10 or 20 Gy of radiation, are shown in Fig. 4. In air-breathing rats, tumor growth was significantly depressed by irradiation with 10 Gy, and irradiation with 20 Gy essentially stopped growth for the 22-day period of observation. When the rats were given carbogen to breathe for 10 min before and during the irradiation, 10 Gy decreased the growth to about one-quarter that of controls (to about one-half that after 10-Gy irradiation of rats breathing air). With 20-Gy irradiation, the tumors of rats breathing carbogen decreased in size until they could no longer be detected after ~10 days, and there was no evidence of recurrence during the 22 days postirradiation through which measurements were made. This compares with rats breathing air where 20 Gy stopped growth at least temporarily but the tumor remained measurable throughout the postirradiation period.
Fig. 4. Effect of breathing carbogen on radiation sensitivity of subcutaneous 9L tumors in rats. Tumors were grown to 6- to 8-mm diameter (~9 days after tumor was injected by trochar; see MATERIALS AND METHODS), divided randomly into 5 groups, and treated as follows: controls, no further treatment; 10 gray (Gy), tumors were treated with 10-Gy irradiation in either animals breathing air or in animals breathing carbogen with irradiated beginning 10 min after initiation of carbogen breathing; 20 Gy, tumors were treated with 20-Gy irradiation in either animals breathing air or in animals breathing carbogen with irradiated beginning 10 min after initiation of carbogen breathing. In all rats, size of tumor was measured at indicated times and tumor volume was calculated (see MATERIALS AND METHODS). Volume of tumor is plotted (ordinate) against no. of days after treatment.
[View Larger Version of this Image (15K GIF file)]

Table  1.   Statistical analysis of effects of 10-gray irradiation on growth of subcutaneous 9L tumors in rats breathing air or carbogen
Treatment Days to Reach 5× Volume Days to Reach 10× Volume
Control 2.9 ± 0.6  3.7 ± 0.7 
10 Gray (breathing air) 3.4 ± 0.2* 5.8 ± 0.3dagger
10 Gray (breathing carbogen) 7.4 ± 0.7dagger 13.6 ± 2.4dagger
Values are means ± SD; n = 6 rats. Carbogen, 5% O2-95% CO2. Tumor volumes were measured as described in MATERIALS AND METHODS. Effect of radiation on rats breathing air was compared with controls (unirradiated), whereas effect of radiation on carbogen-breathing rats was compared with that on rats breathing air. * P < 0.1.  dagger P < 0.001.

When tumor regrowth is followed for a sufficient period of time after irradiation, the growth rate usually returns to control values unless there are complicating events, such as enhanced immune response. In the present experiments, after irradiation the animals were followed for 22 days or until the presence of regrowth was well established but were not followed long enough to establish the maximal rate of regrowth. Table 1 shows a statistical analysis of the time required for tumors subjected to 10-Gy irradiation to grow to 5 and 10 times the size at treatment. The 10-Gy irradiation increased the time to grow five times larger from 2.9 ± 0.6 to 3.4 ± 0.2 days (P < 0.1), and in rats breathing carbogen this increased to 7.4 ± 0.7 days (P < 0.001 compared with 10 Gy in rats breathing air). Similar analysis for the times for the tumors to grow to 10 times treatment volumes shows 10-Gy irradiation increased the time from 3.7 ± 0.7 to 5.8 ± 0.3 days (P < 0.001), whereas in rats breathing carbogen the time increased to 13.6 ± 2.4 days (P < 0.001 compared with 10 Gy in rats breathing air). There was no observable regrowth after 20-Gy irradiation.

DISCUSSION

The measured PO2 values in the vascular system of 9L tumors (1-8 Torr) were substantially below those of normal tissue (25-40 Torr) but were not zero. Thus the tumor tissue was being supplied with oxygen but at PO2 values well below those consistent with healthy normal tissue. Cells require a supply of oxygen at pressures sufficient to allow mitochondrial oxidative phosphorylation to synthesize ATP at the free-energy level ([ATP]/[ADP][Pi] or energy state) necessary for growth and maintenance of the cell. For most normal cells, when the intracellular PO2 falls below ~15 Torr there are compensating metabolic alterations, one of which is a decrease in the cellular energy state (3, 34-36). These alterations in cellular metabolism often include an increase in glycolysis, with ATP production due to metabolism of glucose to lactate partially compensating for the decreased capacity for oxidative phosphorylation. As long as the PO2 does not fall too low, metabolic compensation of the cells is sufficient for viability. Many tumor cells adapt to lower PO2, in part by decreased respiratory capacity and increased glycolytic capacity, achieving a metabolic energy state compatible with growth despite the low PO2. Warburg (31) suggested this characteristic was fundamental to the development of neoplastic tissue, but these alterations in energy metabolism are now generally considered secondary to tumor growth.

The phosphor used in this study was bound to albumin in the blood, and the measurements were of the PO2 in the vessels. The vasculature of tumors is known to be more "leaky" than that of normal tissue, and with longer times the albumin-Pd-porphyrin complex may enter the interstitial space. In the present work, the measurements were begun within a few minutes of probe injection and were completed within ~1 h. If the probe had been exiting from the vessels to the interstitial space in sufficient amounts to interfere with the oxygen measurements, there would have been a systematic and progressive increase in phosphorescence intensity and lifetime over the time of the measurements. Such progressive changes, if present, were small, indicating the phosphorescence measurements were dominated by phosphor in the vasculature.

Oxygen delivery to tissue depends on diffusion of the oxygen from the microvessels to the mitochondria of the cells, where it is consumed. This mass transfer of oxygen is driven by the pressure difference, with the intracellular PO2 (at the mitochondria) being substantially below that of the vessels. This means that in tumors with vascular PO2 values of less than ~10 Torr there are many with PO2 values ~5 Torr. The maps of phosphorescence lifetimes and of PO2 indicate that even tumors <200 µm in diameter have low internal PO2 values relative to the surrounding tissue. Binding of nitroimidazole ([3H]misonidazole) (5, 17) and a 2-nitroimidazole, EF5 (4, 16), has been reported to be enhanced in regions within 9L tumors, consistent with the presence of cells with PO2 values <5 Torr. Similarly, studies of other types of tumors using oxygen electrodes (for review, see Refs. 15, 23, and 26) and estimates of the hypoxic fraction of cells in tumors (2, 4-8) have provided evidence for the presence of low PO2 values in tumors. Oxygen consumption by cells in solid tumors is dependent on the type of cells and their physical environment within the tumor structure. Measurements of the total oxygen consumption of some solid tumors have been reported in which the oxygen consumption was dependent on the rate of delivery (9, 13-14). This increase in respiration as the oxygen delivery increased suggests that these tumors contained oxygen-starved, but viable, cells.

In tumors, poor oxygenation can be a factor in failure of radiation therapy (7, 8, 10). Hyperbaric oxygen has been reported to improve radiotherapy in at least some clinical trials (1, 11, 24). Anemia has been reported to play a significant negative role in the cure rate for carcinoma of the cervix (12). Phosphorescence-quenching measurements show that for subcutaneous tumors grown from 9L cells the PO2 in even small tumors is significantly below that of the surrounding tissue. The signal-to-noise ratio of the measurements is excellent, allowing precise delineation of the limits of the hypoxic regions. When the rats were given 100% oxygen or carbogen to breathe, the PO2 increased in both normal and tumor tissue. In normal tissue, the PO2 increased to well above control (25-35 Torr), achieving levels of 60-80 Torr. The PO2 in tumor tissue also increased, but remained well below that in normal tissue, generally rising to at least 5-15 Torr. The increase in tumor PO2 values was greatest with carbogen breathing, and this was chosen to test for the effect of this increase in PO2 on radiation sensitivity. The time for the tumors irradiated in rats breathing carbogen to grow to a given size was decreased about twofold relative to those irradiated in rats breathing air. This suggests that PO2 values in at least some regions of the tumors were significantly below the O2 half-saturation pressure for the oxygen dependence of radiation sensitivity of ~5 Torr (18). Thus the measured PO2 values, although restricted to the surface layer of the tissue, appear to accurately indicate the PO2 values relevant to radiation sensitivity.

Oxygen-dependent quenching of phosphorescence allows rapid and nondestructive evaluation of the efficacy of treatments designed to alter PO2 values in tumors and other tissues. This will greatly aid development of methods for increasing or decreasing tumor PO2 as an adjunct to treatment. It should be noted that this method is not limited to tumors and can equally aid in critical evaluation of all interventions designed to alter oxygen delivery (blood flow) to any normal and abnormal tissue.

ACKNOWLEDGEMENTS

The authors are indebted to Drs. Cameron Koch and Sydney Evans for encouragement and support.

FOOTNOTES

   This work was supported in part by National Institutes of Health Grants NS-31465 (D. F. Wilson) and CA-44982 (J. Biaglow).

Address for reprint requests: D. F. Wilson, Rm. 426 Anatomy-Chemistry Bldg., Dept. of Biochemistry and Biophysics, Univ. of Pennsylvania, Philadelphia, PA 19104.

Received 12 June 1995; accepted in final form 23 January 1997.

REFERENCES

1. Bush, R. S., R. D. T. Jenkin, W. E. C. Allt, F. A. Beale, H. Bean, A. J. Denko, and J. F. Pringle. Definitive evidence for hypoxic cells influencing cure in cancer therapy. Br. J. Cancer 37, Suppl. III: 302-306, 1978.
2. Chapman, J. D., K. Baer, and J. Lee. Characteristics of metabolism-induced binding of misonidazole to hypoxic mammalian cells. Cancer Res. 43: 1523-1528, 1983 . [Abstract/Free Full Text]
3. Erecinska, M., and D. F. Wilson. Regulation of cellular energy metabolism. J. Membr. Biol. 70: 1-14, 1982 . [Medline]
4. Evans, S. M., C. J. Koch, B. Joiner, W. T. Jenkins, K. M. Laughlin, and E. M. Lord. 2-Nitroimidazole binding for identification of hypoxic cell fraction in cells and tissues of epigastric 9L tumors. Br. J. Cancer 72: 167-174, 1995.
5. Franko, A. J., C. J. Koch, and D. P. J. Boisvert. Distribution of misonidazole adducts in 9L gliosarcoma tumors and spheroids: implications for oxygen distribution. Cancer Res. 52: 3831-3839, 1992 . [Abstract/Free Full Text]
6. Franko, A. J., C. J. Koch, B. M. Garrecht, J. Sharplin, and D. Hughes. Oxygen concentration dependence of binding of misonidazole to rodent and human tumors in vitro. Cancer Res. 47: 5367-5376, 1987 . [Abstract/Free Full Text]
7. Gatenby, R. A., H. B. Kessler, J. S. Rosenblum, L. R. Coia, P. J. Moldofsky, W. H. Hartz, and G. J. Broder. Oxygen distribution in squamous cell carcinoma metastases and its relationship to outcome of radiation therapy. Int. J. Radiat. Oncol. Biol. Phys. 14: 831-838, 1988 . [Medline]
8. Gray, L. H., A. D. Conger, M. Ebert, S. Hornsey, and O. C. A. Scott. Concentration of oxygen dissolved in tissues at time of irradiation as a factor in radiotherapy. Br. J. Radiol. 26: 638-648, 1953.
9. Gullino, P. M., F. H. Grantham, and A. H. Courtney. Utilization of oxygen by transplanted tumors in vivo. Cancer Res. 27: 1020-1030, 1967 . [Abstract/Free Full Text]
10. Hall, E. J. Radiobiology for the Radiologist (3rd ed.). Philadelphia, PA: Lippincott, 1988.
11. Henk, J. M., and C. W. Smith. Radiotherapy and hyperbaric oxygen in head and neck cancer: interim report of the second clinical trial. Lancet 2: 104-105, 1977 . [Medline]
12. Hirst, D. G., J. L. Hazelhurst, and J. M. Brown. The effect of alterations in hematocrit on tumor sensitivity to x-rays. Int. J. Radiat. Biol. 46: 345-354, 1984.
13. Ito, M., A. A. Lammertsma, R. J. S. Wise, S. Bernardi, R. S. J. Frackowiak, J. D. Heather, C. G. McKenzie, D. G. T. Thomas, and T. Jones. Measurement of regional cerebral blood flow and oxygen utilization in patients with cerebral tumors using 15O and positron emission tomography. Neuroradiology 23: 63-74, 1982 . [Medline]
14. Kairento, A. L., G. L. Brownell, D. R. Elmaleh, and M. R. Swartz. Comparative measurements of regional blood flow, oxygen and glucose utilization in soft tissue tumors of rabbit with positron imaging. Br. J. Radiol. 58: 637-643, 1985 . [Abstract/Free Full Text]
15. Kallinowski, F., R. Zander, M. Hockel, and P. Vaupel. Tumor tissue oxygenation as evaluated by computerized PO2-histography. Int. J. Radiat. Oncol. Biol. Phys. 19: 953-961, 1990 . [Medline]
16. Koch, C. J., S. M. Evans, and E. M. Lord. Oxygen dependence of cellular uptake of EF5 2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide: analysis of drug adducts by fluorescent antibodies vs bound radioactivity. Br. J. Cancer. 72: 869-874, 1995 . [Medline]
17. Koch, C. J., C. C. Stobbe, and K. A. Baer. Metabolism induced binding of 14C-misonidazole to hypoxic cells: kinetic dependence on oxygen concentration and misonidazole concentration. Int. J. Radiat. Oncol. Biol. Phys. 10: 1327-1331, 1984 . [Medline]
18. Koch, C. J., C. C. Stobbe, and E. A. Bump. The effect on the Km for radiosensitization at 0°C of thiol depletion by diethylmaleate pretreatment: quantitative differences found using the radiosensitizing agents misonidazole and oxygen. Radiat. Res. 98: 1541-1543, 1984.
19. Leith, J. T., B. S. Schilling, and K. T. Wheeler. Cellular radiosensitivity of a rat brain tumor. Cancer 35: 1545-1550, 1975 . [Medline]
20. Lo, L.-W., C. J. Koch, and D. F. Wilson. Calibration of oxygen dependent quenching of the phosphorescence of Pd-meso-tetra (4-carboxyphenyl) porphine: a phosphor with general application for measuring oxygen concentration in biological systems. Anal. Biochem. 236: 153-160, 1996 . [Medline]
21. Moulder, J. E., and S. Rockwell. Hypoxic fractions of solid tumors: experimental techniques, methods of analysis, and a survey of existing data. Int. J. Radiat. Oncol. Biol. Phys. 10: 695-712, 1984 . [Medline]
22. Rumsey, W. L., J. M. Vanderkooi, and D. F. Wilson. Imaging of phosphorescence: a novel method for measuring oxygen distribution in perfused tissue. Science 241: 1649-1651, 1989.
23. Thews, G., and P. Vaupel. O2 supply conditions in tumor tissue in vivo. Adv. Exp. Med. Biol. 75: 537-546, 1976 . [Medline]
24. Van den Brenk, H. A. Hyperbaric oxygen in radiation therapy. An investigation of dose-effect relationships. Am. J. Roentgenol. 102: 8-26, 1968. [Free Full Text]
25. Vanderkooi, J. M., G. Maniara, T. J. Green, and D. F. Wilson. An optical method for measurement of dioxygen concentration based on quenching of phosphorescence. J. Biol. Chem. 262: 5476-5482, 1987 . [Abstract/Free Full Text]
26. Vaupel, P., H. P. Fortmeyer, S. Runkel, and F. Kallinowski. Blood flow, oxygen consumption, and tissue oxygenation of human breast cancer xenografts in nude rats. Cancer Res. 47: 3496-3503, 1987 . [Abstract/Free Full Text]
27. Vaupel, P., F. Kallinowski, and P. Okunieff. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 49: 6449-6466, 1989 . [Abstract/Free Full Text]
28. Vaupel, P., R. Manz, W. Muller-Kieser, and W. A. Grunewald. Intracapillary HbO2 saturation in malignant tumors during normoxia and hyperoxia. Microvasc. Res. 17: 181-191, 1979 . [Medline]
29. Vinogradov, S. A., L.-W. Lo, W. T. Jenkins, S. M. Evans, C. Koch, and D. F. Wilson. Noninvasive imaging of the distribution of oxygen in tissue in vivo using near infra-red phosphors. Biophys. J. 70: 1609-1617, 1996 . [Medline]
30. Wallen, C. A., S. M. Michaelson, and K. T. Wheeler. Evidence for an unconventional radiosensitivity of rat 9L subcutaneous tumors. Radiat. Res. 84: 529-541, 1980 . [Medline]
31. Warburg, O. On respiratory impairment in cancer cells. Science 124: 269-270, 1956.
32. Wheeler, K. T., and C. A. Wallen. Is cell survival a determinant of the in situ response of 9L tumors? Br. J. Cancer 41, Suppl. IV: 299-303, 1980.
33. Wilson, D. F., and G. J. Cerniglia. Localization of tumors and evaluation of their state of oxygenation by phosphorescence imaging. Cancer Res. 52: 3988-3993, 1992 . [Abstract/Free Full Text]
34. Wilson, D. F., and M. Erecinska. Effect of oxygen concentration on cellular metabolism. Chest 88, Suppl.: 229S-232S, 1982.
35. Wilson, D. F., M. Erecinska, C. Drown, and I. A. Silver. The oxygen dependence of cellular energy metabolism. Arch. Biochem. Biophys. 195: 485-493, 1979 . [Medline]
35. Wilson, D. F., C. S. Owen, and M. Erecinska. Quantitative dependence of mitochondrial oxidative phosphorylation on oxygen concentration: a mathematical model. Arch. Biochem. Biophys. 195: 494-504, 1979 . [Medline]
36. Wilson, D. F., A. Pastuszko, J. E. DiGiacomo, M. Pawlowski, R. Schneiderman, and M. Delivoria-Papadopoulos. Effect of hyperventilation on oxygenation of the brain cortex of newborn piglets. J. Appl. Physiol. 70: 2691-2696, 1991 [Abstract/Free Full Text] .
37. Wilson, D. F., W. L. Rumsey, T. J. Green, and J. M. Vanderkooi. The oxygen dependence of mitochondrial oxidative phosphorylation measured by a new optical method for measuring oxygen concentration. J. Biol. Chem. 263: 2712-2718, 1988 . [Abstract/Free Full Text]
0161-7567/97 $5.00 Copyright © 1997 the American Physiological Society



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cerniglia, G. J.
Right arrow Articles by Biaglow, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cerniglia, G. J.
Right arrow Articles by Biaglow, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online