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. Author manuscript; available in PMC: 2014 Mar 1.
Published in final edited form as: Radiat Res. 2013 Feb 7;179(3):343–351. doi: 10.1667/RR2811.1

Assessment of the Changes in 9L and C6 Glioma pO2 by EPR Oximetry as a Prognostic Indicator of Differential Response to Radiotherapy

Huagang Hou a,d, Sriram P Mupparaju a, Jean P Lariviere a,d, Sassan Hodge c, Jiang Gui b,d, Harold M Swartz a,d, Nadeem Khan a,d,1
PMCID: PMC3633145  NIHMSID: NIHMS460900  PMID: 23391148

Abstract

Tumor hypoxia impedes the outcome of radiotherapy. As the extent of hypoxia in solid tumors varies during the course of radiotherapy, methods that can provide repeated assessment of tumor pO2 such as EPR oximetry may enhance the efficacy of radiotherapy by scheduling irradiations when the tumors are oxygenated. The repeated measurements of tumor pO2 may also identify responders, and thereby facilitate the design of better treatment plans for nonresponding tumors. We have investigated the temporal changes in the ectopic 9L and C6 glioma pO2 irradiated with single radiation doses less than 10 Gy by EPR oximetry. The 9L and C6 tumors were hypoxic with pO2 of approximately 5–9 mmHg. The pO2 of C6 tumors increased significantly with irradiation of 4.8–9.3 Gy. However, no change in the 9L tumor pO2 was observed. The irradiation of the oxygenated C6 tumors with a second dose of 4.8 Gy resulted in a significant delay in growth compared to hypoxic and 2 Gy × 5 treatment groups. The C6 tumors with an increase in pO2 of greater than 50% from the baseline of irradiation with 4.8 Gy (responders) had a significant tumor growth delay compared to nonresponders. These results indicate that the ectopic 9L and C6 tumors responded differently to radiotherapy. We propose that the repeated measurement of the oxygen levels in the tumors during radiotherapy can be used to identify responders and to design tumor oxygen guided treatment plans to improve the outcome.

INTRODUCTION

The imbalance between oxygen supply and consumption often leads to hypoxia in solid tumors, which is believed to compromise the efficacy of radiotherapy and enhance aggressive tumor behavior and metastases (1–5). Consequently, a significant increase in therapeutic outcome may be achieved if tumor hypoxia is minimized by improving the levels of oxygen in solid tumors. Preclinical and clinical investigations using pO2 histograph and assays for hypoxic fractions have shown a substantial change in tumor oxygen after single dose (6, 7) or fractionated radiotherapy (8–12). While some useful information has been obtained in human gliomas by using oxygen electrodes or a fiber-optic probe (13, 14), repeated measurements to assess the time course of the changes in glioma oxygen are not feasible by these methods, because they involve a significant degree of invasiveness, and cannot be used for repeated assessments of tumor pO2 (15).

Several preclinical studies have shown temporal changes in the levels of oxygen of solid tumors during radiotherapy (10, 16–19). We propose that the appropriate schedule of fractions guided by tumor pO2 could enhance therapeutic outcome. Assessment of tumor hypoxia by positron emission tomography (PET) has already shown a great promise in clinical applications (20–22). We have focused on the development of EPR oximetry, which can also provide repeated assessments of average tumor pO2 with minimal perturbation to the microenvironment (23, 24). EPR oximetry has been used extensively for pO2 measurements in animal models (10, 18, 25) and is now being developed for clinical applications (26, 27). Multisite EPR oximetry using magnetic field gradients has further expanded its in vivo application by allowing simultaneous pO2 measurements at 2–4 sites in a tissue of interest (10, 18, 25, 28). In this study, we have investigated the effect of single hypofractionated radiation doses of less than 10 Gy on the ectopic 9L and C6 glioma pO2 by multisite EPR oximetry. The delay in the tumor growth was determined with and without the subsequent fractionation being guided by glioma pO2. Our results indicate that the pO2 of C6 tumors increased significantly with irradiation of 4.8–9.3 Gy. However, no change in the pO2 of the 9L tumors was observed. A significant decrease in the growth of C6 tumors was observed when the subsequent irradiation was scheduled at times of increase in pO2. Therefore, the extent of increase in the pO2 of the C6 tumors during radiotherapy was successfully used to identify responders and nonresponders, which had a significant difference in the tumor growth delay.

MATERIALS AND METHODS

Animals and Tumor Models

All animal procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Dartmouth Medical School (Geisel School of Medicine). The 9L gliomas have a sarcomatous appearance histologically and have been extensively used as a subcutaneous tumor model (29, 30). The C6 gliomas are classified as an astrocytoma with gene expression similar to that of human brain tumors (29, 30). The 9L and C6 tumors are syngeneic to the Fisher and Sprague-Dawley rats, respectively. These tumors were grown in male SCID mice (18–20 g) purchased from Charles River Laboratory (Wilmington, MA) and housed in the animal resource facility at Geisel School of Medicine.

Culture and Inoculation of 9L and C6 Glioma Cells

The 9L and C6 glioma cells were purchased from ATCC (Manassas, VA) and propagated in Dulbecco's Modified Eagle's medium with 4.5 g/L glucose, 1 mM sodium pyruvate, 10% FBS and 1% penicillin-streptomycin. When confluent, the cells were trypsinized and suspended in medium with no serum or additives. The procedure for tumor inoculation has been described previously (10, 18, 25). Briefly, subcutaneous tumors of 6–8 mm in length were obtained approximately 12–14 days after the injection of 100 μl cell suspension containing 4–5 × 105 cells in the left posterior flank of SCID mice.

Implantation of the Oximetry Probe

Lithium phathalocyanine (LiPc) crystals were synthesized in our laboratory and the physicochemical properties of LiPc crystals have been described previously (31). The LiPc crystals have a single sharp EPR line, the width of which is highly sensitive to pO2. It has been shown that the EPR spectra reflect the average partial pressure of oxygen on the surface of each LiPc aggregate and allows the measurements of tumor pO2 using only a few crystals with a weight of approximately 30–50 μg. The volume of each LiPc aggregate is 0.09–0.15 mm3. The procedure for LiPc injection was described previously (10, 18, 25). Briefly, mice were anesthetized with 1.5% isoflurane with 30% O2, and two aggregates of the LiPc crystals were implanted at a depth of 2 mm and a distance of 4 mm into each tumor using 25-gauge needles. A pretreatment (baseline) pO2 of the tumors was measured 24 h after LiPc implantation by multisite EPR oximetry.

Multisite EPR Oximetry

Assessment of tissue pO2 at 2–4 sites both simultaneously and repeatedly by EPR oximetry (referred to as multisite EPR oximetry) has been used in animal models (10, 18, 25, 28). In these experiments, we have assessed pO2 at two sites in each tumor using two implants of LiPc aggregates. The EPR oximetry was performed on an L-band (1,200 MHz) EPR spectrometer using a microwave bridge and an external loop resonator specially designed for in vivo experiments (32). A set of coils capable of generating a magnetic field gradient in the Z-direction with a magnitude up to 3.0 G/cm was used to separate the EPR spectra of the two implants in each tumor (18, 25, 33). The pO2 measured from the two LiPc implants of each tumor were pooled to determine the average pO2 of each tumor.

The spectrometer parameters were: incident microwave power, 2 mW; magnetic field center, 425 gauss; scan range, 2 gauss; modulation frequency, 24 kHz; modulation amplitude was one-third of the EPR line width with scan time of 10 s. The EPR line widths were converted to pO2 using a calibration determined for each batch of LiPc crystals (18, 25, 31).

Experiment Protocol

The mice were assigned randomly into 5 groups for each glioma model: (A and B) control (sham-irradiated on day 1, N9L = 7 and NC6 = 7); (C and D) single dose of 4.8 Gy on day 1 (N9L = 5 and NC6 = 7); (E and F) single dose of 5.7 Gy on day 1 (N9L = 5 and NC6 = 9); (G and H) single dose of 7.0 Gy on day 1 (N9L = 7 and NC6 = 7); and (I and J) single dose of 9.3 Gy on day 1 (N9L = 5 and NC6 = 8). The effect of subsequent irradiation was investigated when the C6 gliomas were oxygenated: (K) 4.8 Gy on day 1 and day 2, NC6 = 24. The results were also compared with standard fractionation and respective control: (L) 2.0 Gy × 5 (NC6 = 7) and (M) control (0 Gy, NC6 = 11). These groups are shown in Table 1.

TABLE 1.

The Number of 9L and C6 Groups with Radiation Dose Used in the Study

Groups Tumor types Doses (Gy) N
A 9L 0 7
B C6 0 7
C 9L 4.8 5
D C6 4.8 7
E 9L 5.7 5
F C6 5.7 9
G 9L 7.0 7
H C6 7.0 7
I 9L 9.3 5
J C6 9.3 8
K C6 4.8 × 2 24
L C6 2.0 × 5 7
M C6 0 11
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For tumor pO2 measurements, the mice were anesthetized (1.5% isoflurane, 30% O2) and positioned between the poles of the EPR magnet. A baseline pO2 was measured for 30 min (day 1) and then the animals were moved to the irradiator bed of a Varian Linear Accelerator (Clinac 2100C, 6 Mev, 6 × 6 cm applicator). The 6 × 6 cm lead applicator had a thickness of 15 mm with a gap (semicircle of 18 mm length and 9 mm radius) in the center to focus the radiation beam on the tumor. An additional lead shield (70 mm length, 60 mm width and 1.5 mm thickness) was used to expose the tumors and further minimize the irradiation of the adjoining normal tissue. The animals were kept anesthetized (1.5% isoflurane, 30% O2) during irradiation. The hypofractionated doses were determined by a calculation based on the linear quadratic formula for cell survival to calculate the “standard equivalent dose (SED)” for planning hypofractionated stereotactic radiotherapy (34, 35). The fractions of 4.8 Gy × 10, 5.7 Gy × 8, 7 Gy × 6 and 9.3 Gy × 4 approximate the biological effect of a 70 Gy (35 fractions of 2 Gy each) dose plan for 9L and C6 tumors (36). The measurements of tumor pO2 were repeated from day 2 to day 5 along with tumor volume measurements by a standard procedure (volume = π/6 × length × width2) from day 1 to day 6. The tumor growth was also followed for up to 14 days to assess tumor growth delay in groups K, L and M.

Physiological Control and Histological Analysis

During EPR measurements, the body temperature of the animals was monitored using a rectal probe and was maintained at 37.0 ± 0.5° using a thermostatically controlled heated pad and a flow of warm air. The animals were kept warm by an electric heating pad during transportation to and from the irradiator. On day 6, the animals were euthanized, and tumors were removed, fixed and sectioned. Microscopic examination (H&E staining) of the tissue around the implanted LiPc deposits was performed to confirm its location in the tumor.

Statistical Analysis

A paired t test was used to determine the statistical significance of changes in pO2 and tumor volume within the group, and an unpaired t test was used to determine the statistical significance between groups. The paired comparison reduces the animal-to-animal heterogeneity and eliminates differences of the baseline pO2. The tumor growth delay was modeled by using an exponential mixed model (37, 38) on the log scale, and estimated by the linear mixed effects (LME) function in the statistical package S-Plus 6.1 (Insightful Inc., Seattle, WA). Assuming an exponential growth, the time to reach 4× the tumor volume of T4TV was computed as T4TV = ln4/a, where “a” is the rate of tumor volume growth obtained from LME. The standard error for T4TV was estimated using the delta method as described by Rice (39). The tests were two-sided, and a change with a P < 0.05 was considered statistically significant. This approach has been used previously to estimate tumor growth (40–42). All data are expressed as mean ± SEM and N is the total number of animals in each group.

RESULTS

Effect of Single-Dose Irradiation on Ectopic Glioma pO2 and Growth

The average pO2 of 9L and C6 tumors after single-dose irradiations with 4.8, 5.7, 7.0 and 9.3 Gy are shown in Figs. 1 and 2, respectively. The 9L tumors were hypoxic with a baseline pO2 of 8.2–8.9 mmHg on day 1 (Fig. 1). The pO2 of the 9L tumors declined with growth over days. Some of the individual 9L tumors showed an increase in tumor pO2. However, no significant change in the average 9L tumor pO2 in each group was observed with irradiation of 4.8–9.3 Gy.

FIG. 1.

FIG. 1

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The ectopic 9L glioma pO2 over days in the groups treated with single doses, panel a: control, 0 Gy, N = 7; panel b: 4.8 Gy, N = 5; panel c: 5.7 Gy, N = 5; panel d: 7.0 Gy, N = 7; panel e: 9.3 Gy, N = 5. (◇) Represents the tumor pO2 recorded from each EPR probe and (◆) represents the mean tumor pO2 within 20 min in the same day. Mean ± SEM, #P < 0.05 vs. baseline tumor pO2 (day 1).

FIG. 2.

FIG. 2

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The C6 glioma pO2 over days in the groups treated, panel a: control, 0 Gy, N = 7; panel b: 4.8 Gy, N = 7; panel c: 5.7 Gy, N = 9; panel d: 7.0 Gy, N = 7; panel e: 9.3 Gy, N = 8. (○) Represents the tumor pO2 recorded from each EPR probe and (●) represent the mean tumor pO2 within 20 min in the same day. Mean ± SEM, #P < 0.05 vs. baseline tumor pO2 (day 1).

The C6 tumors also were hypoxic with a baseline pO2 of 5.4–7.1 mmHg on day 1 (Fig. 2). A significant increase in the pO2 of C6 tumors was observed from day 2 to day 5 of irradiation with single doses of 4.8–9.3 Gy. However, the time to a significant increase in tumor pO2 varied with different doses.

The baseline tumor volumes (day 1) of 9L and C6 tumors were similar between groups (Fig. 3). Irradiation with 4.8–9.3 Gy did not affect the growth of 9L tumors compared to the control group (Fig. 3b). However, the growth of the C6 gliomas were significantly reduced on day 3 to day 6 after single-dose irradiations with 4.8–9.3 Gy (Fig. 3a). No correlation between the baseline tumor pO2 and tumor growth were observed in these experiments.

FIG. 3.

FIG. 3

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The growth of C6 glioma (panel a) and 9L glioma (panel b) over days in the groups treated with (□): control, 0 Gy; (■): 4.8 Gy; (◆): 5.7 Gy; (▲): 7.0 Gy; (●): 9.3 Gy. Mean ± SEM, @P < 0.05, +P < 0.01 vs. control group at the same time point.

Effect of 4.8 Gy × 2 and 2 Gy × 5 on C6 Tumor pO2 and Growth

The lowest dose that led to a significant increase in C6 tumor pO2 on day 2 was 4.8 Gy. Therefore, a subsequent irradiation with 4.8 Gy (group K) was used to investigate the effect on C6 tumor pO2 and growth (Fig. 4). A significant increase in the pO2 of C6 tumor was observed from day 2 to day 4 when irradiated with 4.8 Gy on days 1 and 2. In contrast, no significant increase in the pO2 occurred with irradiation of 2.0 Gy × 5 (Fig. 4, group L, gray diamond symbol).

FIG. 4.

FIG. 4

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Temporal changes in the average C6 glioma pO2 before and after irradiation. (white symbol) 0 Gy (control, N = 11), (black symbol) 4.8 Gy × 2 (day 1, day 2, N = 24) and (gray symbol) 2 Gy × 5 (day 1 to day 5, N = 7). Mean ± SEM, #P < 0.05, &P < 0.01, compared with the baseline pO2 of the same group; @P < 0.05, +P < 0.01, compared with the pO2 of control group on the same day.

No significant differences between the baseline tumor volume of the control and treatment groups were observed (Fig. 5). However, the tumor volume of the 4.8 Gy × 2 (group K) and 2.0 Gy × 5 (group L) significantly decreased from day 5 to day 14 compared to the control (group M). A significant difference in the tumor growth between groups K and L was observed only on day 5 and tumor growth was similar at later time points. The time to reach 4× tumor volume (T4TV) was 10.2 days for the control group, 13.1 and 14.9 days for groups K and L, respectively. The T4TV of group K (4.8 × 2) and L (2.0 Gy × 5) was significantly higher than for group M. However, no significant difference in the T4TV between K and L groups was evident.

FIG. 5.

FIG. 5

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The changes in the C6 glioma growth in (white symbol) 0 Gy (M, N = 11), (black symbol) 4.8 Gy ×; 2 (K, N = 24) and (gray symbol) 2 Gy × 5 (L, N = 7) groups. Inset: Days to reach 4× the original tumor volume (T4TV) of groups M, K and L. The line in bold represents the mean data obtained from individual values on each day. Mean ± SEM, @P < 0.05, +P < 0.01, compared with the control group; †P < 0.05, compared with group L.

To test the prognostic potential of glioma pO2, the animals of group K were separated into subgroups based on the temporal changes in their pO2: K1, C6 gliomas with increase in pO2 on day 2 <50% of baseline (nonresponder), and K2, C6 gliomas with increase in pO2 on day 2 ≥50% of baseline (responder) after 4.8 Gy. The numbers of tumors in the responder and nonresponder groups were 11 and 13, respectively. A significant difference in the tumor pO2 from day 2 to day 5 and growth from day 3 to day 10 was evident between responder and nonresponder groups (Figs. 6 and 7). The T4TV of the responders (11.9 days, K2) was significantly greater than the nonresponders (8.3 days, K1) (Fig. 7, inset). These results indicate that 4.8 Gy irradiation of the oxygenated tumors (responders) on day 2 resulted in a significant growth inhibition compared to hypoxic tumors (nonresponders). Such analyses could not be carried out in group L (2 Gy × 5) due to no change in pO2 during the irradiations.

FIG. 6.

FIG. 6

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The C6 glioma pO2 over days: (●) responders (K2, pO2 on day 2 ≥50% of baseline, N = 11); (○) nonresponders (K1, pO2 on day 2 <50 % of baseline, N = 13). Mean ± SEM, #P < 0.05, &P < 0.01, compared with the baseline pO2 in the same group; @P < 0.05, +P < 0.01, compared with the glioma pO2 of nonresponders on the same day.

FIG. 7.

FIG. 7

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The C6 glioma growth over days: (■) responders (K2, pO2 on day 2 ≥50% of baseline, N 11); (□) nonresponders (K1, pO2 on day 2 <50% of baseline, N = 13). Inset: Number of days to reach 4× of baseline tumor volume (T4TV) of responders and nonresponders. The line in bold represents the mean data obtained from individual values at each time point. Mean ± SEM, @P < 0.05, +P < 0.01, compared with the nonresponders.

Histological Analysis

Histological examination showed fibrotic cells around the LiPc aggregates in the 9L and C6 tumors after 1, 3 and 5 days of implantation (no irradiation group), respectively (Fig. 8). However, some blood cells and inflammatory cells were observed in the tumor sections. These observations represent a typical tissue environment in solid tumors and are unlikely to perturb the tissue pO2 reported by LiPc implants in the tumors. In the normal tissue, we have seen negligible histological changes around LiPc implants (43–45).

FIG. 8.

FIG. 8

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H&E stained sections of the C6 and 9L tumors obtained from the control mice on different days after LiPc implantation. The LiPc crystals (black color) are indicated by long black arrows and blood cells are indicated by short white arrows. The thickness of each section is 5 μm. Magnification: 20×.

DISCUSSION

The data reported here is the continuation of our systematic study to characterize the changes in tumor pO2 after irradiation by EPR oximetry. We have previously reported the effect of a synthetic allosteric modifier of hemoglobin (efaproxiral), a vasodilator, benzyl nicotinate (BN), and carbogen inhalation on the RIF-1 tumor pO2 with the goal to enhance tumor oxygenation and improve treatment efficacy by scheduling irradiations at times of increase in tumor pO2 (10, 18, 25, 46). The results obtained here with ectopic 9L and C6 glioma indicate that these tumors are hypoxic with a pO2 of <10 mmHg, consistent with our earlier observations (47, 48). Cerniglia et al. also has reported ectopic 9L glioma pO2 of less than 8 mmHg in rats using a phosphorescence quenching method (49). Furthermore, a median pO2 of 2 mmHg in the ectopic 9L glioma in the rats has also been observed by Teicher et al. (50, 51). In contrast, orthotopic 9L gliomas in the rats are well oxygenated (36). These results indicate a site specific effect on glioma pO2. Indeed, Wallen et al. suggested a difference in the vasculature between orthotopic and ectopic 9L gliomas as a likely reason for the absence of hypoxia in orthotopic 9L gliomas (52).

The irradiation with single hypofractionated doses of less than 10 Gy did not affect the pO2 of 9L tumors, while a significant increase in the C6 tumor pO2 was observed. A significant increase in the oxygenation of LS174T human colon adenocarcinoma on irradiation with a total dose of more than 10 Gy has been observed by Znati et al. (53). An increase in SCC VII murine tumor pO2 at 6–24 h after irradiation with 10, 15 or 20 Gy was observed by Fuji et al. (54). We have also reported a significant increase in the pO2 of RIF-1 tumors irradiated with 10 Gy (10). Several potential mechanisms have been suggested for the reoxygenation of tumors after irradiation such as reduced oxygen consumption by radiation-damaged cells (55), cell loss leading to tumor shrinkage (56), migration of hypoxic cells to oxygenated areas (57) and improved microcirculation (58).

We observed a significant decrease in the growth of C6 tumors after irradiation, which we did not observe with 9L tumors. This suggested that 9L tumor cells may be relatively radioresistant compared to C6 tumor cells. Indeed, others have shown that the surviving fractions after 2 Gy were indeed significantly higher (71%) for 9L versus 53% for C6 cells respectively (59). As stated above, we observed significant increase in the C6 tumor pO2 and T4TV after irradiation with 4.8 Gy × 2 compared to the control. The T4TV for C6 tumors observed with the 4.8 Gy × 2 was similar to that of the 2 Gy × 5 group. Furthermore, the C6 tumors with an increase in pO2 of more than 50% from the baseline had a significant increase in growth delay compared to those with pO2 less than 50% from the baseline in the 4.8 Gy × 2 (K group). The observed increases in the pO2 of the C6 tumors on day 2 to day 4 are consistent with previous reports (12, 60–62). Fukawa et al. has shown an increase in tumor pO2 as late as day 3 in NFSa fibrosarcomas treated with 25 Gy (63). However, only a few investigators have described the temporal changes in tumor pO2 during fractionated radiotherapy and how this could be used as a prognostic marker during radiotherapy. Our results indicate that the decrease in the C6 tumor growth with treatments of 4.8 Gy × 2 was dependent on the oxygenation status of the tumor after the first irradiation. These results suggest that the temporal changes in tumor pO2 in such cases could indeed potentially be used to identify responders during radiotherapy. We anticipate that this approach could facilitate the design of better therapeutic approaches for nonresponding tumors.

In conclusion, our results demonstrate a glioma specific response to radiotherapy and a significant decrease in the C6 tumor growth when the fractionated radiations of 4.8 Gy × 2 were scheduled at times of increased pO2. These results highlight the importance of tumor pO2 assessment during fractionated radiotherapy and provide evidence that the temporal changes in tumor pO2 can potentially be used to enhance as well as predict therapeutic response. Furthermore, these results also illustrate the ability of EPR oximetry, to make repetitive and noninvasive measurements of pO2 from the same tumors during the course of therapy. EPR oximetry may prove very useful in individualizing the treatment of patients by following the response of tumor pO2 during treatment. We have built clinical EPR spectrometers that will permit us to eventually extend this research into clinical trials (26, 27, 64). EPR oximetry is currently being used to follow the pO2 of superficial tumors such as melanomas and sarcomas in patients undergoing chemoradiation (27). Implantable resonators are currently being pursued for repeated assessment of pO2 in the tumors located at depths of more than 10 mm from the surface (26, 27, 64).

ACKNOWLEDGMENTS

We thank Harriet St. Laurent and Kerry A. Tillson of Radiation Oncology, DHMC for assistance in the use of the radiation facility. This work was supported by NIH grant CA120919 to NK and used the facilities of the EPR Center.

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