In vivo radiation sensitivity of glioblastoma multiforme

Int. J. Radiation Oncology

Pergamon

Biol. Phys., Vol. 32, No. I, pp. 99- 104, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0360-3016/95 $9.50 + .oo

0360-3016(94)00494-3

l

Biology Original Contribution IN VZVO RADIATION ALPHONSE MICHAEL

SENSITIVITY

OF GLIOBLASTOMA

MULTIFORME

WILFRIED BUDACH, M.D.,” TAGHIAN, M.D., PH.D., *+ WILLEM DUBOIS, M.D.,’ BAUMANN, M.D.: JILL FREEMAN, B.S.* AND HERMAN SUIT, M.D., D.PHIL.*

*Edwin L. Steele Laboratory of Radiation Biology, Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; +Department of Radiation Oncology, Boston University Medical Center, Boston, MA; $Department of Radiation Therapy, Dr. Daniel den Hoed Kliniek, Groene filledjek 301, 3075 EA Rotterdam, The Netherlands; “Department of Radiation Therapy, University of Essen, Hufelandstr.55, D-45 122, Essen, Germany; “Department of Radiation Therapy, University Hospital Eppendorf, Martinistr. 52, 20246 Hamburg, Germany Purpose: Human glioblastoma (GBM) is one of the most resistant tumors to radiation. In previous reports, we have demonstrated a wide range of radiation sensitivity of GBM in v&o; that is, SF, values of 0.2 to 0.8. The great sensitivity of some of the cell lines is not in accord with the almost invariably fatal clinical outcome of patients with GBM. The sensitivity of cells in vitro pertains to cells cultured in optimal nutritional conditions. The TCD, (the radiation dose necessary to control 50% of the tumors locally) determined in lab animals is analogous to the use of radiation with curative intent in clinical radiation oncology. The aim of the present study was (a) to evaluate the sensitivity of GBM in viva relative to that of other tumor types and (b) assess the relationship between the single dose TCD, of the xenografts and the sensitivity of the corresponding cell lines in vitro. Methods and Materials: The TCD, assay was used to study twelve human tumor lines. Four previously published values were added. A total of 10 GBM, 4 squamous cell carcinoma (SCC), 1 soft tissue sarcoma (STS), and 1 cancer colon (CC) are included in the analysis. For further suppression of the residual immune system, all the animals received 6 Gy whole-body irradiation 1 day before transplantation. Local tumor irradiations were given as a single dose, under conditions of clamp hypoxia using a Cs irradiator. Results: The TCDSO values for the 10 GBM xenografts varied between 32.5 and 75.2 Gy, with an average of 47.2 2 13.1 Gy. The TCD, values for the SCC were similar to those of the GBM and ranged from 40.7 and 54.4 Gy, with a mean of 46.8 5 6.4. The difference between the average TCD, of GBM and SCC was not significant. The STS and CC xenografts bad TCD, values of 46.0 and 49.2 Gy, respectively. No correlation was found between the TCDso in vivo and the SF2 or D,, in vitro. Conclusions: Our data on GBM xenografts showed a wide range of sensitivities to single dose irradiation in uivo, which does not correlate with the almost invariably fatal clinical outcome of these patients. No correlation was observed between the TCD, in vivo and the in vitro SFJD,, of the corresponding ce]] lines. Our in vivo and in vitro data on GBM suggest that radiation sensitivity alone does not explain the cause of the poor clinical response of GBM to radiation, and other factors could contribute to this response. Glioblastoma multiforme,

In vivo, Xenografts,

Nude mice, Radiation sensitivity, TCD,.

INTRODUCTION

suggested to be one of the major causes of the poor clinical results of GBM (7). In our laboratory, we investigated the role of radiation sensitivity as a determinant of failure of GBM to respond to treatment. In a previous report (21), we evaluated the intrinsic radiation sensitivity of 21 cell lines derived from mahg-

Glioblastoma multiforme (GBM) is known to be among the most resistant tumors to radiation. Virtually, no patients with GBM survive 5 years following treatment. The intrinsic radiation sensitivity of these tumors has been

This paper was presented at the 35th Annual Meeting of the American Society for Therapeutic Radiology and Oncology, New Orleans, LA, 11- 15 October 1993.

DHHS Grant CA13311 awardedby the National CancerInstitute, Departmentof Health andHumanServices,the “Associa-

Dr. H.D. Suit is the Andres Soriano Oncology, Harvard Medical School.

and by the Phillip Foundation.The authorswould like to thank

tion pour la Recherche

Professor of Radiation

Contre le Cancer (ARC)”

in France,

R. Sedlacek, E. Rose, and T. Ebert for their vaIuable collaboration and suggestions. They also thank S. Wendt, S. Marshall,

Reprint requeststo: AlphonseTaghian,M.D., Ph.D., Edwin L. Steele Laboratory of Radiation Biology, Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA

P. Morris, H. Phamfor animalcaretaking,and P. McNally for her excellent assistancein the preparationof this manuscript. Accepted for publication 9 September1994.

02114. Acknowledgements-This work was supported in part by 99

100

I. J. Radiation Oncology 0 Biology 0 Physics

Volume 32, Number 1, 1995

Table 1. Designation, histology, and origin of the human tumor xenografts used in this study Designation of the tumor

Histology

HGL4 HGL9 HGLl1 HGL21 MMCl

Glioblastoma Glioblastoma Glioblastoma Glioblastoma Glioblastoma

D54MG U251MG T98G

Glioblastoma Multiforme Glioblastoma Multiforme Glioblastoma Multifonne

scc21

Head and neck squamous cell carcinoma Head and neck squamous cell carcinoma

MGH

Soft tissue sarcoma Cancer colon

Dr. Little, Boston ATCC

SQ20B STS-26T HCTlS

Multiforme Multiforme Multiforme Multiforme Multiforme

Origin MGH* MGH MGH MGH Dr. Komblith (NYC’) Dr. Bigner (Duke) Dr. Bigner (Duke) ATCd

Dr. Little, Boston

* Massachusetts General Hospital; +New York City; ‘American type culture collection.

nant gliomas. The parameter surviving fraction at 2 Gy (SF,) was used to describe the intrinsic radiation sensitivity in vitro. Unexpectedly, the SF* of these cell lines in vitro varied widely, and some malignant glioma cell lines were quite radiation sensitive. These findings do not correlate with the almost invariably fatal outcome of patients with GBM; however, similar results were shown by other investigators (I, 14). In a review on malignant gliomas cell lines (25), no correlation was found between the survival of the patients and the corresponding SF, values. These data suggested that the intrinsic radiation sensitivity in vitro may not be the major determinant of the poor clinical results of GBM in vivo. The following phase of our in vitro research has been to evaluate the recovery capacity of cell lines derived from GBM (24). Here again, a wide range of recovery capacity for GBM was found, suggesting that this factor alone cannot explain the clinical results of GBM. One possible reason for this incongruity may be that under in vitro conditions, the cells are in near optimal metabolic and nutritional environment, which might not be the case in tumors in vivo. The in vivo model might be closer to the clinical situation than the in vitro one. The tumor control dose 50% or TCDSO (the radiation dose necessary to control 50% of the tumors locally) determined in lab animals is analogous to the use of radiation with curative intent in clinical radiation oncology. We

therefore studied GBM xenografts into nude mice and we used the TCDsO as an endpoint. The aim of this study is: (a) to evaluate the sensitivity of GBM in vivo relative to that of squamous cell carcinoma (SCC), soft tissue sarcoma, and cancer colon xenografts; and (b) assess the relationship between the single dose TCD50 of the xenografts and the sensitivity of the corresponding cell lines (SF? and Do) measured in vitro.

METHODS Animals Eight to ten week-old,

AND MATERIALS male and female NCr/Sed

(nd

nu) nude mice from our specific defined flora animal colony (17) were used in all experiments. The guidelines for the humane treatment of animals in this study have been approved by the Subcommittee on Research Animal Care Office of Laboratory Animal Resources in the Massachusetts General Hospital. To minimize the residual immune response, all animals received 6 Gy whole-body irradiation 24 h prior to tumor transplantation

Gammacell

(22, 27), using a

‘37Cs unit (0.8 Gy/min).

Tumor transplantation

Twelve human tumors were used: eight of them were GBM, two head and neck SCC, one STS, and one CC. From the eight GBM, four were derived from patients at the Massachusetts General Hospital (MGH) and established in our laboratory. Table 1 shows the designation, histology, and origin of each tumor. The establishment and transplantation of the tumors were previously described (3, 4). Briefly, lo7 to lo* tumor cells from the in vitro cell culture (21) were injected into the axilla of nude mice. The resulting tumors were passaged to the back of a second group of animals. The tumors from these animals were excised, cleaned from the necrotic debris, cut into chunks, and transplanted into the right hindleg of the nude mice. Eighty to ninety animals were used per experiment. Table 2. The designation, histology, TCDso, SF, and D, values of the human tumor xenografts and the

corresponding cell lines Designation of the tumor

TCDSO (GY)

Glioblastoma multifotme 33.2 HGL4 47.8 HGL9 HGLi 1 32.5 43.0 HGL21 MMCl 52.9 37.1 D54MG T98G 43.3 44.2 U251MG A7 60.9$ U-87MG 75.2’ Squamous cell carcinoma 42.6 scc2 1 SQ2OB 49.6 40.71: HSCC6 54.4* FaDu Soft tissue sarcoma 46.0 STS-26T Colon cancer 49.2 HCT15

95% Confidence interval

SF2

Do

(28.1-39.3) (42.5-52.3) (27.7-38.0) (31.9-48.8) (48.0-58.0) (30.0-46.0) (35.0-53.0) (38.0-51.0) (56.9-65.4) (70.7-80.0)

0.39* 0.56* 0.64* 0.54* 0.52* 0.49* 0.74* 0.40* 0.59* 0.50*

1.58* 1.39* 1.70* 1.55* 1.57* 1.05* 1.61’ 1.39* 1.56* 2.25*

(30.9-58.8) (43.1-57.0) (37.5-44.5) (52.2-56.7)

0.65’ 0.62* 0.40’

1.46’ 1.50% 1.00*

(41.4-51.0)

0.38$

1.06”

(42.4-57.0)

0.751

2.00+

* Taghian et al. (Reprinted, with permission, from IJROBP 23:55-62; 1992); ‘Taghian et al. (unpublished data); ‘Suit et al. (Reprinted, with permission, from IJROBP 18:365-373; 1990); “Ruka et al. (unpublished data).

In vivo

radiation sensitivity of

Irradiation The tumor diameters were measured three times per week before, and one to two times per week after, irradiation. The tumor volume was determined by the formula 7r16 x a x b2, where a is the longer and b the perpendicular shorter tumor axis. Irradiation was delivered to the tumor when its volume reached 120 mm3. The animals were randomized into control or the different treatment groups. Single dose irradiations were applied using 8 to 10 graded dose levels ranging from 15 to 85 Gy, using a specially designed Cesium irradiator (12), featuring parallel opposed fields, at a dose rate of 6.5 to 7 Gy/min. The animals were anesthetized with pentobarbital (0.05 mg/g body weight) administered intraperitoneally, and a heavy clamp was applied to stop the blood flow 3 min before and during the irradiation. Six to twelve animals were assigned for each dose level. Determination of the TCD50 The mice were followed up for a period of 6-12 months; that is, until the TCDSo values reached a plateau of a minimum of 3 months. The dose necessary to control 50% of the tumors locally and their 95% confidence interval were calculated by regression analysis on a logit-log grid, correcting for censored animals (19, 26). RESULTS In vivo data Table 2 shows the TCDSO values of 16 human tumor xenografts in nude mice together with SF2 and Do of the corresponding cell lines in vitro. The irradiation in vivo was performed using single doses under clamp hypoxia. The dose response in vitro was studied by a colony formation assay under aerobic conditions using exponentially growing cells (21). Four of the TCDso values and most of the survival parameters in vitro have been published previously (20, 21). The TCD50 experiments of these four tumors were performed by different investigators in the same laboratory using the same techniques under the same conditions. The TCDSO values for the 10 GBM xenografts varied between 32.5 and 75.2 Gy with an average of 47.2 2 13.1 Gy. The TCDSo values for the SCC varied between 40.7 and 54.4 Gy with an average of 46.8 + 6.4. The difference between the average TCDso of GBM and that of SCC was not significant. The soft tissue sarcoma and cancer of colon xenografts had a TCDSo of 46.0 and 49.2 Gy, respectively. Figure 1 shows the cumulative frequency of the TCDso values of the GBMs, illustrating the wide range of in viva sensitivity with a coefficient of variation of 28%. Figure 2 compares this distribution of TCDSO values of GBM with the results of xenografts of SCC, STS, and CC. There is an extensive overlap between the distributions. Correlation between the in vivo and in vitro data Figure 3 compares the TCDSO values with the values of SF, (Fig. 3a) and Do (Fig. 3b) of the corresponding

GBM

0 A. TAGHIAN

101

et al.

cell lines. All tumors listed in Table 1, except SCC21 (which could not be established in vitro), are included. There was no correlation between the intrinsic radiation sensitivity measured in vitro, and described either by SF2 (Fig. 3a) or by the Do (Fig. 3b) and the in vivo sensitivity determined by the TCDSO. DISCUSSION Several reports have demonstrated that the resistance of GBMs to clinical radiation therapy does not correspond to their intrinsic radiation sensitivity (1, 14, 21, 25). We extended these studies and investigated the radiation response of human GBM in nude mice relative to the response of SCC and STS. In the experiments reported here, influence of the hypoxic tumor cell fraction, cell cycle effects, reoxygenation, and repopulation of clonogenic tumor cells during treatment were minimized by the use of single dose irradiations under clamp hypoxia. Under such highly standardized experimental conditions, tumor control doses may be quantitatively described by the number of tumor rescuing units and their radiation sensitivity in vivo (4, 10, 19). Therefore, if there were systematic differences in these parameters between GBM and other tumor entities, it should be expected that this is reflected in higher TCDsO values. However, our results demonstrate a wide overlapping of the distribution of TCDso values between GBM, SCC, CC, and STS (Fig. 2), suggesting that much more complex mechanisms are involved in the radiation resistance of GBM in patients. One concern in local control experiments using human tumor xenografts in nude mice is that the residual immune response reaction of the hosts may affect the results in some tumors (5, 11, 15, 18, 22, 27). Even if such influences of the residual immune response reaction cannot be completely ruled out, it appears for the following reasons unlikely that such an effect had important impact on the results reported here: 1. All animals have been whole-body irradiated with 6 Gy, which has been shown to significantly reduce the

1.0 -

0.0, 30

s 40

r 50

TCDSO

I 60

.

I 70

f 80

(Gy)

Fig. 1. The cumulative frequency of TCDSo of glioblastoma multiforme

xenografts

into nude mice.

102

I. J. RadiationOncology l Biology 0 Physics

residual immune response in nude mice (5, 15, 18, 22, 27). 2. Using quantitative transplantation assays, we have demonstrated that severe combined immune deficient (SCID) mice (8) were significantly less immune reactive against human xenografts than nude mice (22). Therefore, if the residual immune response of nude mice effects the TCDso values, we would expect that the TCDSO values of the human tumors in SCID mice are higher than the values for the same tumors implanted in nude mice. However, in a series of experiments reported from our laboratory, Budach er al. (6) showed that the TCDSO values of eight out of nine human and murine tumors implanted into SCID mice were not higher than in nude mice. These experiments included three human tumors that were also investigated in the present study. The TCD,o values of two of these (HGL9 and STS-26T) were not significantly different in SCID mice and nude mice, whereas the TCDso values of cancer of the colon (HCTIS) were 49.2 Gy and 58 Gy in nude and in SCID mice, respectively. In experiments on FaDu tumors irradiated at different sizes, no difference of TCDSO values was observed between normal and whole-body irradiated nude mice (4). Therefore, at least for three tumors included in the present study, any influence of residual immune response reaction on local control can be excluded, and extrapolating from these results, an important impact appears unlikely for most of the remaining tumors. 3. There is no basis to assume that the residual immune response reaction of nude mice effects only GBM. Because we determined TCDSO values for GBM relative to that of SCC, CC, and STS using exactly the same experimental conditions, it is expected that any unwarranted influence of the residual immune response reaction on TCDSO values will impact the distribution of TCDSO values in all entities to about the same degree.

I

70

60

I

0

50

.

40

; 0

; :

1

8

Fig. 2. The TCDso of the 10 glioblastoma multiforme (GBM) compared to that of four squamous cell carcinoma (XC), one soft tissue sarcoma (STS), and one cancer colon (CC) xenografts into nude mice.

Volume 32, Number 1, 1995

0.3

0.4

0.5

0.6

0.7

0.8

SF2

-_ I (b) 70-

22 g 60. t n 8 so-

0

0

0 .O

8

l om

40~,

0 . .

0.5

GBM see STSKC

) 1.0

.

l o

) . 1.5

. , 2.0

, 2.5

DO

Fig. 3. The correlation between the in vivo sensitivity described by the TCD, and the in vitro sensitivity described by the SF, (a) or the Do (b) of 15 tumor lines: 10 glioblastoma multifonne (GBM), 3 squamous cell carcinoma (KC) (SCC21 did not have a corresponding in vitro cell line), 1 soft tissue sarcoma (STS), and 1 cancer colon (CC) xenografts. One possible explanation for the lack of correlation of radiobiological parameters with the clinical results in GBM is that both the in vitro, as well as the in vivo, experiments were performed outside the normal environment of GBM; that is, outside the brain. It could be argued that the microenvironment in the central nervous system might alter the radiation sensitivity of these tumors. It has been shown by several authors (23,27) that there is a significant enhancement of the transplantability of murine and human tumors in the brain in comparison to that of the SC tissue. The cause is not well known; however, some authors (2, 16) have suggested that the brain might be a partly immune-privileged site. Fiedler (9) demonstrated that when a human colon cancer was implanted orthotopically into the colon of nude mice, it behaved in a similar way to the original tumor in terms of liver metastasis. If this is true for brain tumors, GBM implanted into the brain of nude mice might be more radiation resistant than when implanted in the SC tissue. Another possibility to explain our findings is that GBM tumors per se are not as resistant as we thought, or the radiation sensitivity of GBM is not the major determinant

103

In vivo radiation sensitivity of GBM 0 A. TAGHIAN et al.

of the poor clinical outcome of these tumors. Other factors might be more important than the radiosensitivity in the response of GBM to radiation. It is not excluded that the radiation resistance of GBM is at least in part caused by radiobiological mechanisms that were not evaluated in our experiments using single dose irradiation under clamp hypoxic conditions. These parameters include repair of radiation damage between fractions, redistribution in cell cycle, reoxygenation, and repopulation of clonogenic tumor cells during treatment. This notion is supported by the observation reported from our laboratory by Baumann et al. (3) that the TCDSo, after 30 fractions under normal blood flow conditions, was higher in four of five malig-

nant gliomas than in two SCC xenografts. Another important parameter that might be implicated in the response of GBM to radiation is hypoxia. Rumpling er al. (13) have used oxygen electrodes to measure the pOz values of GBM in 10 patients. They have shown that the pooled median pOz value was 7.4 mmHg. They also found a high percentage (28%) of low pOz values indicating a radiobiologically significant hypoxic component in all GBM studied. This could have a strong influence on radiosensitivity. Our finding that the radiation response in vivo did not correlate with the intrinsic radiation sensitivity in vitro (Fig. 3) adds to previously reported results demonstrating that determinations of SF, in vitro are not sufficient to explain the clinical radiation resistance of GBM (1, 14,

21) or the response of human tumor xenografts treated with fractionated radiation therapy (3). It also suggests that one single factor might not be enough to predict the tumor response to radiation. This is in agreement with Gerweck et al. (10). They found that no single parameter (clonogenic fraction, hypoxic fraction, or intrinsic radio-

sensitivity) correlated with the observed TCDSo under aerobic or hypoxic conditions. However, when considered in combination, clonogenic fraction and intrinsic radiosensitivity predicted the rank-order of TCDSO with a significant degree of accuracy. However, it may be speculated that differences in the microenvironment between in vitro cell culture, SC tissues in nude mice, and the tumor site in patients, as well as racliobiological parame-

ters not evaluated by in vitro radiation cell survival assays, contribute to this observation. CONCLUSION Our results on GBM xenografts showed a wide range

of sensitivities to single dose irradiation in vivo, which does not correlate with the invariably fatal clinical outcome of these patients. There was no correlation between the TCDSO in vivo and the SF& measured in vitro. Our in vivo and in vitro data on GBM (21, 25) suggest that radiation sensitivity alone does not explain the cause of the poor clinical responseof GBM to radiation, and other

factors could contribute to this response.

REFERENCES 1. Allalunis-Turner, J.; Barron, G.; Day, R.; Fulton, D.; Urtasun, R. Radiosensitivity testing of human primary brain tumor specimens. Int. J. Radiat.Oncol. Biol. Phys.23:339343; 1992. 2. Barker C. F.; Billingham,R. E. Immunologicallyprivileged sites.Adv. Immunol. 51-54; 1977. 3. Baumann,M.; duBois,W.; Pu, A.; Freeman,J.; Suit, H. D. Responseof xenografts of humanmalignantgliomasand

squamous cell carcinomas to fractionated irradiation. Int. J. Radiat. Oncol. Biol. Phys. 23:803-809; 1992. 4. Baumann,M.; DuBois, W.; Suit, H. Responseof a human

squamous cell carcinoma xenografts to irradiation at differemssizes:Relationshipof clonogeniccells, cellular radiation sensitivity in vim, and tumor rescuingunits. Radiat. Res. 123:325-330; 1990. 5. Baumann,M.; Pu, A.; DuBois,W.; Suit, H. D. Quantitative evaluation of the effects of cotransplantationof heavily irradiatedhumantumor cells and of different immunosuppressivemeasureson the xenotransplantabilityof a human squamous cell carcinomainto athymic nudemice. Contrib. Oncol. 42:98- 107; 1992. 6. Budach, W.; Taghian, A.; Freeman, J.; Gioioso, D.; Suit, H. The impact of stromal sensitivity on the radiation responseof tumors.J. Natl. CancerInst. 85988-993; 1993. 7. Davis, L. Presidentialaddress:Malignant glioma-A nemesiswhich requiresclinical andbasicinvestigationin radiation oncology. Int. J. Radiat. Oncol. Biol. Phys. 16:1355-

1365; 1989. 8. Dorshkind,K.; Pollack, S. B.; Bosma,M. J.; Phillips, R. A. Natural killer (NK) cell are presentin mice with severe

combined immunodeficiency(SCID). J. Immunol. 134: 3798-3801; 1985. 9. Fiedler, I. Orthotopic implantationof humancolon carcinoma into nude mice provides a valuable model for the biology and therapy of metastasis. CancerMetastasisRev. 10:229-243; 1991. 10. Gerweck, L. E.; Zaidi, S.; Zietman, A. Multivariate determinantsof radiocurability. I: Prediction of singlefraction tumor control doses.Int. J. Radiat. Oncol. Biol. Phys. 29:57-66; 1994. 11. Heberman,R. B. Naturalcell mediatedcytotoxicity in nude mice. In: Fogh, J.; Giovanella,B. C., eds.The NudeMouse in ExperimentalandClinical Research.Vol. 2. New York: Academic Press,Inc.; 1982:79-93. 12. Hranitzky, E.; Almond, P.; Suit, H. D.; Moore, E. A cesium137 irradiator for small laboratory animals. Radiology 107641-644; 1973. 13. Rampling,R.; Cmickshank,G.; Lewis, A.; Fitzsimmons, S.; Workman, P. Direct measurement of pOZdistribution and bioreductive enzymes in human malignant brain tumors.Int. J. Radiat. Oncol. Biol. Phys. 29:427-431; 1994. 14. Ramsay, J.; Ward, R.; Bleehen, N. Radiosensitivity testing of humanmalignantgliomas.Int. J. Radiat. Oncol. Biol. Phys. 24:625-680; 1992. 15. Rofstad, E. K. Local tumor control following singledose irradiation of humanmelanomaxenografts:Relationshipto cellular radiosensitivity and influence of an immuneresponseby the athymic mouse.CancerRes.49:3163-3 167; 1989. 16. ScheinbergL.; EdelmanF.; Levy, W. A. Is the brain “an immunologicallyprivileged site?” 1. Studiesbasedon in-

104

17.

18.

19.

20.

21.

I. I. RadiationOncology 0 Biology 0 Physics tracerebral tumor homotransplantation and isotransplantation to sensitized hosts. Arch. Neurol. 11:248-264; 1964. Sedlacek, R.; Orcutt, P.; Suit, H.; Rose, E. A flexible barrier at cage level for existing colonies: Production and maintenance of a limited stable anaerobic flora in a closed inbred mouse colony. In: Sasaki, S., ed. Recent Advances in Germ Free Research. Tokyo: Tokai University Press; 1981:6569. Silobrcic, E.; Zietman, A.; Ramsay, J.; Suit, H.; Sedlacek, R. Residual immunity of athymic NCr/Sed nude mice and the xenotransplantation of human tumors. Int. J. Cancer 45325-333; 1990. Suit, H.; Shalek, R.; Wette, R. Radiation response of mouse mammary carcinoma evaluated in terms of cellular radiation sensitivity. In: Shalek, R. J., et aZ., eds. Cellular Radiation Biology. Baltimore: Williams and Wilkins; 1965:514530. Suit, H. D.; Zietman, A.; Tomkinson, K.; Ramsay, J.; Gerweek, L.; Sedlacek, R. S. Radiation response of xenografts of a human squamous cell carcinoma and a glioblastoma multiforme: A progress report. Int. J. Radiat. Oncol. Biol. Phys. 18:365-373; 1990. Taghian, A.; Suit, H.; Pardo, F.; Gioioso, D.; Tomkinson, K.; Dubois, W.; Gerweck, L. In vitro intrinsic radiation sensitivity of glioblastomamultiforme. Int. J. Radiat. Oncol. Biol. Phys. 23:55-62; 1992.

Volume 32, Number 1, 1995 22. Taghian,A.; Budach,W.; Zietmann,A.; Freeman,J.; Gioioso, D.; Ruka, W.; Suit, H. D. Quantitative comparison betweenthe transplantabilityof humanand murinetumors into the SC tissueof NCr/Sed (nu/nu) nude and (SCID) mice. CancerRes.53:5012-5017; 1993. 23. Taghian,A.; Budach,W.; Zietmann,A.; Freeman,J.; Gioioso, D.; Suit, H. D. Quantitative comparisonbetweenthe transplantabilityof humanandmurinetumorsinto the brain of NCr/Sed (nu/nu) nude and (SCID) mice. Cancer Res. 53:5018-5021; 1993. 24. Taghian, A.; Gioioso, D.; Budach, W.; Suit, H. D. Splitdose recovery of glioblastomamultiforme. Radiat. Res. 134:16-21; 1993. 25. Taghian,A.; Ramsay,J.; Allalunis-Turner,J.; Budach,W.; Gioioso,D.; Pardo,F.; Okunieff, P.; Bleehen,N.; Urtasun, R.; Suit, H. D. Intrinsic radiationsensitivity may not be the majordeterminantof the poor clinical outcomeof glioblastomamultiforme.Int. J. Radiat.Oncol. Biol. Phys. 25:243249; 1993. 26. Walker, A.; Suit, H. Assessmentof local tumor control usingcensoredtumor responsedata. Int. J. Radiat. Oncol. Biol. Phys. 9:383-386; 1983. 27. ZietmanA.; Suit H. D.; Ramsay,J.; Silobrcic, V.; Sedlacek, R. Quantitative studieson the transplantabilityof murine and humantumors into the brain and SC tissuesof NCr/ Sed nudemice. CancerRes.48:6510-6516; 1988.