Int. J Radiation Oncology Bml. Phys., Vol. Printed in the U.S.A. All rights reserved.
19, pp. 197-202
Copyright
0360-3016/90 $3.00 + NJ 0 1990 Pergamon Press plc
??Oncology Intelligence
HYPOXIC FRACTIONS OF HUMAN TUMORS XENOGRAFTED INTO MICE: A REVIEW SARA ROCKWELL, PH.D.’ AND JOHN E. MOULDER, PH.D.~ ‘Department of Therapeutic Radiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 065 10; and 2Department of Radiation Oncology, Medical College of Wisconsin, 8700 West Wisconsin Ave., Milwaukee, WI 53226
Transplantedtumors in rats and mice have been used extensively in radiobiology studies examining the biological basis and therapeutic implications of hypoxia in solid tumors, and in studies evaluating new agents and regimens which may circumvent the radioprotective effects of hypoxiccells. This use of rodent tumors assumes that data obtained using rapidly-growing transplanted tumors arising in inbred rodents can be extrapolated meaningfully to the treatment of primary and/or metastatic lesions in heterozygous humans. The studies reported here examine one facet of this critical assumption by comparing the hypoxic fractions of transplanted rodent tumors with those of human tumor cell lines xenografted into immune deficient mice. No significant differences were found between the hypoxic fractions of the xenografts and those of mouse tumors. This findingeliminatesseveral possible bases for predicting that the oxygenation of human tumors might be systematically different from that of the rodent tumors often used in preclinical radiobiology studies, and supports the hypothesis that human tumors contain hypoxic cells which may be critical in determining their response to therapy. Solid tumor, Human tumor xenograft, Hypoxia, Radiotherapy, Tumor oxygenation.
INTRODUmION Transplanted
rodent
tumors
ficient rodents are similar to the hypoxic fractions of the experimental rodent tumors generally used in radiobio-
in mice and rats have been
logical studies. There are some theoretical bases to expect that there might be systematic differences between the oxygenation of xenografts with malignant cells of human origin and that of transplanted rodent tumors.
used extensively in radiobiology studies examining the biological basis and therapeutic implications of hypoxia in solid tumors. Because direct evidence for the presence of hypoxic cells in human tumors remains limited (1, 4, 6, 13, 17,259, rodent studies have provided the preclinical background supporting clinical trials with such modalities as hyperbaric oxygen, perfluorochemical emulsions, hypoxic cell radiosensitizers, hypoxia-selective cytotoxins, and drugs which modulate tumor blood flow (4, 14). The use of animal tumor data to develop agents and regimens for clinical trials rests on the hypothesis that data obtained with rapidly-growing transplanted tumors in highly inbred rodents can be extrapolated meaningfully to clinical situations involving primary and/or metastatic lesions in heterozygous humans (14). This critical assumption is not straightforward or trivial. It is, therefore, important to examine in detail any and all data which may be useful in assessing the validity and limitations of this extrapolation. This review examines one aspect of this hypothesis, by asking whether the effective radiobiological hypoxic fractions of human tumors xenografted into immune de-
We were able to find 27 data sets that could be used to calculate hypoxic fractions for human tumor cell lines xenografted into immune-deficient mice. The studies
Reprint requests to: Sara Rockwell, Ph.D. Acknowledgements-The authors thank Drs. Guichard, Rofstad, Zeitman, and Suit and their collaborators for sharing with us and allowing us to quote their unpublished data and data still in press. This paper was presented at the 1989 Meeting of the
American Society for Therapeutic Radiology and Oncology, San Francisco, October 4, 1989. This research was supported in part by Grant CA-35215 from the National Cancer Institute and Grant PDT- 145 from the American Cancer Society Accepted for publication 24 January 1990.
METHODS AND MATERIALS Radiobiological data sets which could be used to calculate hypoxic fractions were found through a review of the literature. Additional data sets, as yet unpublished or in press, were graciously provided to us by the investigators. To ensure that the data from these different sources were analyzed in a uniform manner, all dose-response data were re-analyzed using the model-based analyses described in detail previously (15, 16). RESULTS
197
198
1. J. Radiation Oncology 0 Biology 0 Physics Table I. Hypoxic fractions
of human
July 1990, Volume 19, Number I tumor xenografts
in immune
deficient
mice
Tumor
Histology
Size’
Assay system’
Tdi
Do, ti’
Ref.
Bell Bell EE EE EF GE GE Mall MF Nail Nail Nal I Nal Ih Nail VN VN HCTR HRT18 HT29 No name* MLS MLS OWI OWI U87 FaDu HSCC6
Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Cola-rectal Colo-rectal Colo-rectal Pancreatic Ovarian Ovarian Ovarian Ovarian Glioma Squamous Ca Squamous Ca
7.4 6.2 9.0 9.1 9.1 8.5 9.1 7.3 9.1 5.5 6.7 6.6 6.6 7.0 8.5 9.1 6.3 5.7 8.2 6.5 7.3 15.5 7.3 15.5 6.0 6.0 6.0
PSC PSC PSC PSC PSC PSC PSC PSC PSC PSC GD CD GD PSC PSC PSC PSC PSC PSC PSC PSC PSC PSC PSC Cure Cure Cure
4-5 4-5 6 4.8 17.4 4.2 3.4 4-5 16.4 4.3 I2 I2 II 6.2 6 5 I 3-4 8 I7 3 4 3.6 5.5 12.3
5.9 7.0 2.3 2.6 3.5 3.2 3.2 4.2 3.3 3.4 2.9 2.9 2.9 3.3 2.9 2.5 2.7 3.7 3.0 3.9 4.1 3.7 3.7 7.0 4.1 4.1
2 9 I8.20,2 I 20 20 5,19 20 2 20 IO 7.12 8.12 8 I I,12 5,19 20 9 9 9
-
Hypoxic fraction’
22 22 22 22 24 24 24
’ Mean diameter (in mm) estimated from size data given by authors. 2 PSC: Paired survival curves; CD: growth delay: Cure: paired TCDstr’s. 3 Td: Tumor volume doubling time, in days. 4 Do, h: Do for the hypoxic tumor cells. calculated as described in References I5 and 16. 5 Hypoxic fraction is given as percent of total tumor cells. Values in parentheses are 95% confidence ’ Tumors grown in nude rats.
40 41 6.8 6.8 23 32 32 60 39 85 77 85 91 84 I4 13 27 14 76 20 8.8 28 I6 I8 0.19 0.64 19
(26-63) (32-54) (4.2-l 1) (4.9-9.4) (16-23) (22-42) (23-46) (42-88) (27-57) (67-100) (19-100) (50-100) (45-100) (69-100)’ (8.3-23) (8.5-21) (18-41) (6.7-30) (25-100) (16-23) (6.8-l 1) (21-26) (13-21) (I 3-24) (0.02-I .8) (0.018-1.9) (4.4-63)
limits.
’ Hypoxic fraction estimated from survival data at 20 Gy. ’ Implanted intramuscularly.
yielding these data are summarized on Table 1. All of these experiments were performed using established cell lines, rather than primary biopsy material or early passage serially transplanted xenografts. In all but one experiment, tumors were grown in nude mice; one Nal 1 study used nude rats. Most tumors were grown subcutaneously: in four studies the site was not specified but is probably subcutaneous on the basis of the methodology used by the same investigators in other studies. One study used intramuscular tumors. A variety of histologic types are included in the analyses. The spectrum of histologies among the xenografts is very different from the spectrum observed previously ( 15, 16) among the transplanted rodent tumors generally used in radiobiology studies. The majority of the rodent tumors were anaplastic sarcomas or mammary carcinomas. In contrast, the majority of the xenografts were melanomas. with other clinically resistant neoplasms (cola-rectal, pancreatic cancer, glioma, ovarian cancer) comprising the great majority of the tumors studied. This probably reflects the desire of the investigators to elucidate the mechanism
underlying the resistance of these neoplasms by looking for unusual cellular radioresistance or unusually high hypoxic fractions. Although paired survival curve analyses are most common, three TCDSO analyses and two growth delay studies are also included in the data base. Because several different approaches to calculating hypoxic fractions had been taken in the analyses which had been published by the investigators, we re-analyzed all of these data sets using the techniques described and used in our previous analyses of hypoxic fractions in rodent tumors ( 15, 16). These calculated hypoxic fractions are shown on Table 1. The distributions of hypoxic fractions of the xenografts and of transplanted rodent tumors of similar size are compared on Figure 1. This comparison included only studies using paired survival curves to avoid any possible bias arising from a variation in hypoxic fraction with the assay system used ( 15, 16). The hypoxic fractions for the xenografts, shown on Table 1, are in the same range as those found previously for transplanted rodent tumors of similar sizes, although the mean hypoxic fractions for xe-
Hypoxia in human tumor xenografts 0 S. ROCKWELL AND
199
J. E. MOULDER
0.4 s .2
0.3 -
ZL 2 a
0.2 0.1
-
1
.I
10
100
Hypoxic Fraction (%)
.lI
Fig. 1. Comparison of the hypoxic fractions of human tumor xenografts and of transplanted rodent tumor lines. Data for a specific tumor were considered statistically compatible with a stated hypoxic fraction ifthat value lay within the 95% confidence limits determined in our analysis. Only data obtained using paired survival curve assays of SC, im, and id tumors with diameters of 5-10 mm were included in this comparison. ----: Data for xenografts; detailed data are presented in Table 1. -: Data for studies using transplanted rodent tumors of similar sizes; the rodent tumor data are presented and discussed in References 15 and 16.
nografts (2 1%) is slightly above that for transplanted rodent tumors ( 14%). Comparison of the two distributions
of hypoxic fractions using the Mann-Whitney U-test (26) revealed no significant differences in the distributions, whether the comparison was based on all assays (p = 0. lo), or only on excision assays (p = 0.11). We examined the relationship between the tumor volume doubling time (Td) and the hypoxic fraction (Fig. 2). Our previous analyses ( 15, 16) with transplanted rodent tumors had revealed no variation in the hypoxic fraction with Td; however, that analysis lacked statistical power, because the majority of the tumors had very short dou-
100
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Tumor Diameter (mm) Fig. 3. Relationship between tumor size (mean diameter in mm) and hypoxic fraction for human tumor xenografts. Data are replotted from Table 1. Each point represents a single excision (0) or in situ assay (0). Individual data points represent tumors studied in only 1 experiment. Points connected by lines represent tumors (A OWI; w MLS) studied at two sizes. The solid line is the best fit to the data when OWI and MLS data are excluded.
bling times, and only a handful of tumors had doubling times 2 1 week. In contrast, the median doubling time for the xenografts was 5.5 days and eight had Td > 1 week. The longer doubling times of the xenografts produced a statistically more powerful analysis of the relationship between tumor growth rate and hypoxic fraction. It might be predicted that slowly-growing tumors would have lower hypoxic fractions, because the vasculature would be better able to “keep up” with the growth of the tumor. The data shown on Figure 2 reveal no relationship between Td and hypoxic fraction for excision assays (r = 0.10, p > 0.20). For the four tumors studied using in situ assays, the hypoxic fractions are larger for slowly-growing tumors than for rapidly-growing tumors (r = .97, p = ~0.05). We also examined the relationship between tumor size and hypoxic fraction (Fig. 3). This analysis is more limited than our previous analyses with transplanted rodent tumors, as the range of tumor volumes examined for the xenografts is much smaller (all xenografts were studied at macroscopic sizes, >5 mm diameter) and as there are only a few studies in which the same tumor was deliberately examined at different sizes. These data reveal no significant trend of hypoxic fraction with tumor size (r = 0.09, p > 0.20). This finding is in contrast to the increase in the hypoxic fraction with size seen in the more extensive and statistically more powerful analysis of the rodent tumor data (Fig. 4).
Tumor Volume Doubling Time (days) Fig. 2. Relationship between the tumor volume doubling time (Td) and the hypoxic fraction of human tumor xenografts. Each point represents a single determination made using an excision (0) or in situ (0) assay. The solid line is the best fit to the excision data, the dashed line is the best fit to the in situ data. Data are shown in detail in Table 1.
DISCUSSION Human tumor xenografts are being used increasingly as models in experimental cancer therapy, in the hope that the tumor lines may maintain the inherent charac-
200
July 1990, Volume 19, Number 1
1. J. Radiation Oncology 0 Biology 0 Physics
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Hypox~c Fraction (O/o)
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Fig. 4. Relationship between tumor size (mean diameter in mm) and hypoxic fraction of transplanted rodent tumors in studies examining changes in hypoxia with size in individual tumor systems. Points are hypoxic fractions, with 95% confidence limits; solid symbols represent SCand im tumors, open symbols represent pulmonary nodules. Data for rodent tumors were obtained over a wider range of sizes than were the data for xenografts shown on Figure 3. These data reveal a change in hypoxic fraction with tumor volume which is not seen in the xenograft data shown on Figure 3. (Reprinted from reference 14, with permission).
teristics of the specific human cancers from which they were derived, and therefore might be useful in developing agents or regimens which will be effective against specific human diseases (19, 23). In the study reported here, we collected and reviewed radiobiological data which could be used to calculate hypoxic fractions for human tumor xenografts. This was done to assess whether there were any systematic differences between the hypoxic fractions of the xenografts and those of the transplanted rodent tumors generally used in laboratory studies of tumor hypoxia. Such systematic differences might occur, for example, if human tumor cells were intrinsically more sensitive than rodent tumor cells to the cytotoxic effects of severe hypoxia, low pH, or glucose deprivation; in this case the proportions of viable, hypoxic cells in the xenografts might be much smaller than those of transplanted rodent tumors. Similarly, if human and rodent cells differed significantly in their abilities to induce neovascularization, the hypoxic fractions of the human and rodent tumors might differ systematically. The different origins and relatively well-differentiated phenotypes of xenografts (relative to serially transplanted rodent tumors) could conceivably produce systematic differences between the oxygenation of the xenografts and the rodent tumors. In this respect, it was disappointing to discover that virtually all these xenograft studies were performed using relatively poorly differentiated tumors derived from established cell lines; studies with early passage xenografts or well-differentiated xenograft lines would be valuable. It is interesting that both the hypoxic fractions and the values for Do,h calculated for the xenografts spanned the same ranges observed for rodent tumors, especially because the xenografts were
over-represented by tumors of notable clinical radioresistance. On the whole, the xenografts surveyed grew more slowly than the transplanted rodent tumors included in other previous surveys. It has been postulated that slowly-growing neoplasms might be better oxygenated than rapidlygrowing tumors, because the vasculature might be better able to match the growth of the tumor and thereby maintain an adequately functional vascular bed. However, we found no evidence to support this hypothesis. There appeared to be no correlation between the tumor volume doubling time and hypoxic fraction of the xenografts, except for the four systems assayed in situ, where the hypoxic fraction was actually higher for slowly-growing tumors than for the rapidly-growing tumors. We found no evidence that the slowly-growing xenografts were better oxygenated than rapidly-growing mouse and rat tumors. Our data provided no evidence for a significant or systematic difference between the oxygenation of xenografts and rodent tumors. It should be recalled that there are limitations to the use of xenografts to model oxygenation in human tumors. First, the stroma of the xenografts is of mouse origin; the growth rate and characteristics of the vascular bed therefore will have murine rather than human characteristics. Moreover, the host of a xenograft is, of course, a mouse. To the extent that the oxygenation of the tumors depends on the characteristics of the binding and release of O2 by hemoglobin, the blood pressure, the arterial p02, etc., xenografts in mice and transplanted rodent tumors in mice will be similar. Third, the xenografts, like rodent tumor lines, are transplanted tumors. If the relationship between a transplanted tumor and its host differs from the relationship between a primary,
Hypoxia in human tumor xenografts 0 S. RCCKWELL
Never Transplanted
) lncreasmg Transplant Generations
Culture Adapted
Fig. 5. Effect of the transplantation history of mouse mammary tumors on their hypoxic fractions. Hypoxic fractions were estimated from radiation response data for autochthenous tumors growing in their hosts of origin (X), for tumors transplanted once (0) 2-5 times (0), lo-100 times (V v), > 100 times (OH), and for cell culture adapted tumor lines (A A). Open symbols represent in situ assays, closed symbols represent excision assays. These data provide no evidence that the transplantation history of the tumors influences their hypoxic fractions or that there is any difference in the hypoxic fractions of primary and transplanted tumors. (Reprinted from reference 16, with permission).
AND
J. E. MOULDER
201
spontaneous neoplasm and its host in a way which influences the oxygenation of the tumor cells, transplantation will affect xenografts as well as rodent tumor lines. However, our previous analyses (Fig. 5) comparing the hypoxic fractions of autochthenous mouse mammary tumors, early transplant passages of mouse mammary tumors, long-passaged mouse mammary tumor lines, and cell culture-adapted mouse mammary tumor lines ( 16) provided no evidence that the transplantation history of rodent tumors influenced their oxygenation. Thus, transplantation per se does not appear to influence tumor oxygenation. The similarity of the hypoxic fractions of human tumor xenografts and transplanted rodent tumors eliminates some possible bases for postulating a systematic difference between the hypoxic fractions of human cancers and those of the transplanted rodent tumors often used in radiobiology studies. These studies provide an additional reason to expect that studies using transplanted rodent tumors to study tumor oxygenation, reoxygenation, and the modulation of tumor oxygenation during radiotherapy can be extrapolated to predict and interpret the situation in human tumors.
REFERENCES 1 Cater, D. B.; Silver, I. A. Quantitative measurements of oxygen tension in normal tissues and in the tumours of patients before and after radiotherapy. Acta Radiol. 53:233256; 1960. 2. Chavaudra, N.; Guichard, M.; Malaise, E. P. Hypoxic fraction and repair of potentially lethal radiation damage in two human melanomas transplanted into nude mice. Radiat. Res. 8856-68; 198 I _ 3. Courtenay, V. D.; Smith, 1. E.; Peckham, M. J.; Steel, of human tumour G. G. In vitro and in vivo radiosensitivity cells obtained from a pancreatic carcinoma xenograft. Nature 263:771-772; 1976. 4. Dische, S. Chemical sensitizers for hypoxic cells: a decade of experience in clinical radiotherapy. Radiother. Oncol. 3: 97-l 15; 1985. 5. Flaten, T. P.; Rofstad, E. K.; Brustad, T. Radiation response oftwo human malignant melanomas grown in athymic nude mice. Europ. J. Cancer 17:527-532; 1981. 6. Gatenby, R. A.; Coia, L. R.; Richter, M. P.; Katz, H.; Moldofsky, P. J.; Engstrom, P.; Brown, D. Q.; Brookland, R.; Broder, G. J. Oxygen tension in human tumors: in vivo mapping using CT-guided probes. Radiol. 1562 11-2 14; 1985. 7. Guichard, M.; Chavaudra, N.; Malaise, E. P. Tumour growth delay and cell survival as endpoints for studying the fraction of hypoxic cells in a human melanoma transplanted into nude mice. Int. J. Radiat. Biol. 38:349-353; 1980. 8. Guichard, M.; Chavaudra, N.; Malaise, E. P. Is the proportion of hypoxic cells in a solid tumor dependent on the host (mouse or rat)? Bull. Cancer 76:367-371; 1989. 9. Guichard, M.; Dertinger, H.; Malaise, E. P. Radiosensitivity of four human tumor xenografts. Influence of hypoxia and cell-cell contact. Radiat. Res. 95:602-609; 1983. 10. Guichard, M.; Gosse, C.; Malaise, E. P. Survival curve of a
I1
12.
13.
14.
15.
16. 17.
18.
19. 20.
human melanoma in nude mice. J. Natl. Cancer Inst. 58: 1665-1669; 1977. Guichard, M.; Malaise, E. P. Radiosensitivity of Nal 1 human melanoma transplanted into nude mice: Repair, reoxygenation and dose fractionation. Int. J. Radiat. Oncol. Biol. Phys. 8:1005-1010; 1982. Lehnert, S. and Guichard, M. Radioresistance of human tumor xenograhs: Possible Mechanisms. NC1 Mono. 6:205209; 1988. Moore, J. V.; Hasleton, P. S.; Buckley, C. H. Tumour cords in 52 human bronchial and cervical squamous cell carcinomas: inferences for their cellular kinetics and radiobiology. Br. J. Cancer 51:407-413; 1985. Moulder, J. E.; Dutreix, J.; Rockwell, S.; Siemann, D. W. Applicability of animal tumor data to cancer therapy in humans. Int. J. Radiat. Oncol. Biol. Phys. 14:913-927; 1988. Moulder, J. E.; Rockwell, S. 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. Moulder, J. E.; Rockwell, S. Tumor hypoxia: its impact on cancer therapy. Cancer Metastasis Rev: 5:3 13-34 1; 1987. Mueller-Kheser, W.; Vaupel, P.; Manz, R.; Schmidseder, R. Intracapillary oxyhemoglobin saturation in malignant tumors in humans. Int. J. Radiat. Oncol. Biol. Phys. 7: 13971404; 1981. Rofstad, E. K. Radiation response of the cells of a human malignant melanoma xenograft. Effect of hypoxic cell radiosensitizers. Radiat. Res. 87:670-683; 198 1. Rofstad, E. K. Human tumor xenografts in radiotherapeutic research. Radiother. Oncol. 3:35-46; 1985. Rofstad, E. K. Hypoxia and reoxygenation in human melanoma xenografts. Int. J. Radiat. Oncol. Biol. Phys. 17:8 I89; 1989.
202
I. J. Radiation Oncology 0 Biology 0 Physics
21. Rofstad, E. K.; Brustad. T. The radiosensitizing effect of metronidazole and misonidazole (Ro-07-0582) on a human malignant melanoma grown in the athymic nude mouse. Br. J. Radiol. 5 I:38 I-386; 1978. 22. Rofstad, E. K.; Fenton, B. M.; Sutherland, R. M. Intracapillary HbOz saturations in murine tumours and human tumour xenografts measured by cryospectrophotometry: relationship to tumour volume, tumour pH and fraction of radiobiologically hypoxic cells. Br. J. Cancer 57:494-502; 1988. 23. Steel, G. G.; Peckham, M. J. Human tumour xenografts: a critical appraisal. Br. J. Cancer (Suppl. IV): 133- 14 1; 1980.
July 1990, Volume 19. Number 1 24. Suit, H. D.; Zeitman, A.; Tomkinson, K.; Ramsay, J.; Gerweek. L.; Sedlacek. R. 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. 25. Urtasun, R. C.; Chapman, J. D.; Raleigh, J. A.; Franko, A. J.; Koch, C. J. Binding of ‘H-misonidazole to solid human tumors as a measure of tumor hypoxia. Int. J. Radiat. Oncol. Biol. Phys. 12: 1263- 1267: 1986. 26. Zar. J. H. Biostatistical analysis, 2nd edition. Cliffs, N.J.: Prentice-Hall. Inc.: 1984.
Englewood
19, pp. 197-202
Copyright
0360-3016/90 $3.00 + NJ 0 1990 Pergamon Press plc
??Oncology Intelligence
HYPOXIC FRACTIONS OF HUMAN TUMORS XENOGRAFTED INTO MICE: A REVIEW SARA ROCKWELL, PH.D.’ AND JOHN E. MOULDER, PH.D.~ ‘Department of Therapeutic Radiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 065 10; and 2Department of Radiation Oncology, Medical College of Wisconsin, 8700 West Wisconsin Ave., Milwaukee, WI 53226
Transplantedtumors in rats and mice have been used extensively in radiobiology studies examining the biological basis and therapeutic implications of hypoxia in solid tumors, and in studies evaluating new agents and regimens which may circumvent the radioprotective effects of hypoxiccells. This use of rodent tumors assumes that data obtained using rapidly-growing transplanted tumors arising in inbred rodents can be extrapolated meaningfully to the treatment of primary and/or metastatic lesions in heterozygous humans. The studies reported here examine one facet of this critical assumption by comparing the hypoxic fractions of transplanted rodent tumors with those of human tumor cell lines xenografted into immune deficient mice. No significant differences were found between the hypoxic fractions of the xenografts and those of mouse tumors. This findingeliminatesseveral possible bases for predicting that the oxygenation of human tumors might be systematically different from that of the rodent tumors often used in preclinical radiobiology studies, and supports the hypothesis that human tumors contain hypoxic cells which may be critical in determining their response to therapy. Solid tumor, Human tumor xenograft, Hypoxia, Radiotherapy, Tumor oxygenation.
INTRODUmION Transplanted
rodent
tumors
ficient rodents are similar to the hypoxic fractions of the experimental rodent tumors generally used in radiobio-
in mice and rats have been
logical studies. There are some theoretical bases to expect that there might be systematic differences between the oxygenation of xenografts with malignant cells of human origin and that of transplanted rodent tumors.
used extensively in radiobiology studies examining the biological basis and therapeutic implications of hypoxia in solid tumors. Because direct evidence for the presence of hypoxic cells in human tumors remains limited (1, 4, 6, 13, 17,259, rodent studies have provided the preclinical background supporting clinical trials with such modalities as hyperbaric oxygen, perfluorochemical emulsions, hypoxic cell radiosensitizers, hypoxia-selective cytotoxins, and drugs which modulate tumor blood flow (4, 14). The use of animal tumor data to develop agents and regimens for clinical trials rests on the hypothesis that data obtained with rapidly-growing transplanted tumors in highly inbred rodents can be extrapolated meaningfully to clinical situations involving primary and/or metastatic lesions in heterozygous humans (14). This critical assumption is not straightforward or trivial. It is, therefore, important to examine in detail any and all data which may be useful in assessing the validity and limitations of this extrapolation. This review examines one aspect of this hypothesis, by asking whether the effective radiobiological hypoxic fractions of human tumors xenografted into immune de-
We were able to find 27 data sets that could be used to calculate hypoxic fractions for human tumor cell lines xenografted into immune-deficient mice. The studies
Reprint requests to: Sara Rockwell, Ph.D. Acknowledgements-The authors thank Drs. Guichard, Rofstad, Zeitman, and Suit and their collaborators for sharing with us and allowing us to quote their unpublished data and data still in press. This paper was presented at the 1989 Meeting of the
American Society for Therapeutic Radiology and Oncology, San Francisco, October 4, 1989. This research was supported in part by Grant CA-35215 from the National Cancer Institute and Grant PDT- 145 from the American Cancer Society Accepted for publication 24 January 1990.
METHODS AND MATERIALS Radiobiological data sets which could be used to calculate hypoxic fractions were found through a review of the literature. Additional data sets, as yet unpublished or in press, were graciously provided to us by the investigators. To ensure that the data from these different sources were analyzed in a uniform manner, all dose-response data were re-analyzed using the model-based analyses described in detail previously (15, 16). RESULTS
197
198
1. J. Radiation Oncology 0 Biology 0 Physics Table I. Hypoxic fractions
of human
July 1990, Volume 19, Number I tumor xenografts
in immune
deficient
mice
Tumor
Histology
Size’
Assay system’
Tdi
Do, ti’
Ref.
Bell Bell EE EE EF GE GE Mall MF Nail Nail Nal I Nal Ih Nail VN VN HCTR HRT18 HT29 No name* MLS MLS OWI OWI U87 FaDu HSCC6
Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Cola-rectal Colo-rectal Colo-rectal Pancreatic Ovarian Ovarian Ovarian Ovarian Glioma Squamous Ca Squamous Ca
7.4 6.2 9.0 9.1 9.1 8.5 9.1 7.3 9.1 5.5 6.7 6.6 6.6 7.0 8.5 9.1 6.3 5.7 8.2 6.5 7.3 15.5 7.3 15.5 6.0 6.0 6.0
PSC PSC PSC PSC PSC PSC PSC PSC PSC PSC GD CD GD PSC PSC PSC PSC PSC PSC PSC PSC PSC PSC PSC Cure Cure Cure
4-5 4-5 6 4.8 17.4 4.2 3.4 4-5 16.4 4.3 I2 I2 II 6.2 6 5 I 3-4 8 I7 3 4 3.6 5.5 12.3
5.9 7.0 2.3 2.6 3.5 3.2 3.2 4.2 3.3 3.4 2.9 2.9 2.9 3.3 2.9 2.5 2.7 3.7 3.0 3.9 4.1 3.7 3.7 7.0 4.1 4.1
2 9 I8.20,2 I 20 20 5,19 20 2 20 IO 7.12 8.12 8 I I,12 5,19 20 9 9 9
-
Hypoxic fraction’
22 22 22 22 24 24 24
’ Mean diameter (in mm) estimated from size data given by authors. 2 PSC: Paired survival curves; CD: growth delay: Cure: paired TCDstr’s. 3 Td: Tumor volume doubling time, in days. 4 Do, h: Do for the hypoxic tumor cells. calculated as described in References I5 and 16. 5 Hypoxic fraction is given as percent of total tumor cells. Values in parentheses are 95% confidence ’ Tumors grown in nude rats.
40 41 6.8 6.8 23 32 32 60 39 85 77 85 91 84 I4 13 27 14 76 20 8.8 28 I6 I8 0.19 0.64 19
(26-63) (32-54) (4.2-l 1) (4.9-9.4) (16-23) (22-42) (23-46) (42-88) (27-57) (67-100) (19-100) (50-100) (45-100) (69-100)’ (8.3-23) (8.5-21) (18-41) (6.7-30) (25-100) (16-23) (6.8-l 1) (21-26) (13-21) (I 3-24) (0.02-I .8) (0.018-1.9) (4.4-63)
limits.
’ Hypoxic fraction estimated from survival data at 20 Gy. ’ Implanted intramuscularly.
yielding these data are summarized on Table 1. All of these experiments were performed using established cell lines, rather than primary biopsy material or early passage serially transplanted xenografts. In all but one experiment, tumors were grown in nude mice; one Nal 1 study used nude rats. Most tumors were grown subcutaneously: in four studies the site was not specified but is probably subcutaneous on the basis of the methodology used by the same investigators in other studies. One study used intramuscular tumors. A variety of histologic types are included in the analyses. The spectrum of histologies among the xenografts is very different from the spectrum observed previously ( 15, 16) among the transplanted rodent tumors generally used in radiobiology studies. The majority of the rodent tumors were anaplastic sarcomas or mammary carcinomas. In contrast, the majority of the xenografts were melanomas. with other clinically resistant neoplasms (cola-rectal, pancreatic cancer, glioma, ovarian cancer) comprising the great majority of the tumors studied. This probably reflects the desire of the investigators to elucidate the mechanism
underlying the resistance of these neoplasms by looking for unusual cellular radioresistance or unusually high hypoxic fractions. Although paired survival curve analyses are most common, three TCDSO analyses and two growth delay studies are also included in the data base. Because several different approaches to calculating hypoxic fractions had been taken in the analyses which had been published by the investigators, we re-analyzed all of these data sets using the techniques described and used in our previous analyses of hypoxic fractions in rodent tumors ( 15, 16). These calculated hypoxic fractions are shown on Table 1. The distributions of hypoxic fractions of the xenografts and of transplanted rodent tumors of similar size are compared on Figure 1. This comparison included only studies using paired survival curves to avoid any possible bias arising from a variation in hypoxic fraction with the assay system used ( 15, 16). The hypoxic fractions for the xenografts, shown on Table 1, are in the same range as those found previously for transplanted rodent tumors of similar sizes, although the mean hypoxic fractions for xe-
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nografts (2 1%) is slightly above that for transplanted rodent tumors ( 14%). Comparison of the two distributions
of hypoxic fractions using the Mann-Whitney U-test (26) revealed no significant differences in the distributions, whether the comparison was based on all assays (p = 0. lo), or only on excision assays (p = 0.11). We examined the relationship between the tumor volume doubling time (Td) and the hypoxic fraction (Fig. 2). Our previous analyses ( 15, 16) with transplanted rodent tumors had revealed no variation in the hypoxic fraction with Td; however, that analysis lacked statistical power, because the majority of the tumors had very short dou-
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Tumor Diameter (mm) Fig. 3. Relationship between tumor size (mean diameter in mm) and hypoxic fraction for human tumor xenografts. Data are replotted from Table 1. Each point represents a single excision (0) or in situ assay (0). Individual data points represent tumors studied in only 1 experiment. Points connected by lines represent tumors (A OWI; w MLS) studied at two sizes. The solid line is the best fit to the data when OWI and MLS data are excluded.
bling times, and only a handful of tumors had doubling times 2 1 week. In contrast, the median doubling time for the xenografts was 5.5 days and eight had Td > 1 week. The longer doubling times of the xenografts produced a statistically more powerful analysis of the relationship between tumor growth rate and hypoxic fraction. It might be predicted that slowly-growing tumors would have lower hypoxic fractions, because the vasculature would be better able to “keep up” with the growth of the tumor. The data shown on Figure 2 reveal no relationship between Td and hypoxic fraction for excision assays (r = 0.10, p > 0.20). For the four tumors studied using in situ assays, the hypoxic fractions are larger for slowly-growing tumors than for rapidly-growing tumors (r = .97, p = ~0.05). We also examined the relationship between tumor size and hypoxic fraction (Fig. 3). This analysis is more limited than our previous analyses with transplanted rodent tumors, as the range of tumor volumes examined for the xenografts is much smaller (all xenografts were studied at macroscopic sizes, >5 mm diameter) and as there are only a few studies in which the same tumor was deliberately examined at different sizes. These data reveal no significant trend of hypoxic fraction with tumor size (r = 0.09, p > 0.20). This finding is in contrast to the increase in the hypoxic fraction with size seen in the more extensive and statistically more powerful analysis of the rodent tumor data (Fig. 4).
Tumor Volume Doubling Time (days) Fig. 2. Relationship between the tumor volume doubling time (Td) and the hypoxic fraction of human tumor xenografts. Each point represents a single determination made using an excision (0) or in situ (0) assay. The solid line is the best fit to the excision data, the dashed line is the best fit to the in situ data. Data are shown in detail in Table 1.
DISCUSSION Human tumor xenografts are being used increasingly as models in experimental cancer therapy, in the hope that the tumor lines may maintain the inherent charac-
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Fig. 4. Relationship between tumor size (mean diameter in mm) and hypoxic fraction of transplanted rodent tumors in studies examining changes in hypoxia with size in individual tumor systems. Points are hypoxic fractions, with 95% confidence limits; solid symbols represent SCand im tumors, open symbols represent pulmonary nodules. Data for rodent tumors were obtained over a wider range of sizes than were the data for xenografts shown on Figure 3. These data reveal a change in hypoxic fraction with tumor volume which is not seen in the xenograft data shown on Figure 3. (Reprinted from reference 14, with permission).
teristics of the specific human cancers from which they were derived, and therefore might be useful in developing agents or regimens which will be effective against specific human diseases (19, 23). In the study reported here, we collected and reviewed radiobiological data which could be used to calculate hypoxic fractions for human tumor xenografts. This was done to assess whether there were any systematic differences between the hypoxic fractions of the xenografts and those of the transplanted rodent tumors generally used in laboratory studies of tumor hypoxia. Such systematic differences might occur, for example, if human tumor cells were intrinsically more sensitive than rodent tumor cells to the cytotoxic effects of severe hypoxia, low pH, or glucose deprivation; in this case the proportions of viable, hypoxic cells in the xenografts might be much smaller than those of transplanted rodent tumors. Similarly, if human and rodent cells differed significantly in their abilities to induce neovascularization, the hypoxic fractions of the human and rodent tumors might differ systematically. The different origins and relatively well-differentiated phenotypes of xenografts (relative to serially transplanted rodent tumors) could conceivably produce systematic differences between the oxygenation of the xenografts and the rodent tumors. In this respect, it was disappointing to discover that virtually all these xenograft studies were performed using relatively poorly differentiated tumors derived from established cell lines; studies with early passage xenografts or well-differentiated xenograft lines would be valuable. It is interesting that both the hypoxic fractions and the values for Do,h calculated for the xenografts spanned the same ranges observed for rodent tumors, especially because the xenografts were
over-represented by tumors of notable clinical radioresistance. On the whole, the xenografts surveyed grew more slowly than the transplanted rodent tumors included in other previous surveys. It has been postulated that slowly-growing neoplasms might be better oxygenated than rapidlygrowing tumors, because the vasculature might be better able to match the growth of the tumor and thereby maintain an adequately functional vascular bed. However, we found no evidence to support this hypothesis. There appeared to be no correlation between the tumor volume doubling time and hypoxic fraction of the xenografts, except for the four systems assayed in situ, where the hypoxic fraction was actually higher for slowly-growing tumors than for the rapidly-growing tumors. We found no evidence that the slowly-growing xenografts were better oxygenated than rapidly-growing mouse and rat tumors. Our data provided no evidence for a significant or systematic difference between the oxygenation of xenografts and rodent tumors. It should be recalled that there are limitations to the use of xenografts to model oxygenation in human tumors. First, the stroma of the xenografts is of mouse origin; the growth rate and characteristics of the vascular bed therefore will have murine rather than human characteristics. Moreover, the host of a xenograft is, of course, a mouse. To the extent that the oxygenation of the tumors depends on the characteristics of the binding and release of O2 by hemoglobin, the blood pressure, the arterial p02, etc., xenografts in mice and transplanted rodent tumors in mice will be similar. Third, the xenografts, like rodent tumor lines, are transplanted tumors. If the relationship between a transplanted tumor and its host differs from the relationship between a primary,
Hypoxia in human tumor xenografts 0 S. RCCKWELL
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Fig. 5. Effect of the transplantation history of mouse mammary tumors on their hypoxic fractions. Hypoxic fractions were estimated from radiation response data for autochthenous tumors growing in their hosts of origin (X), for tumors transplanted once (0) 2-5 times (0), lo-100 times (V v), > 100 times (OH), and for cell culture adapted tumor lines (A A). Open symbols represent in situ assays, closed symbols represent excision assays. These data provide no evidence that the transplantation history of the tumors influences their hypoxic fractions or that there is any difference in the hypoxic fractions of primary and transplanted tumors. (Reprinted from reference 16, with permission).
AND
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spontaneous neoplasm and its host in a way which influences the oxygenation of the tumor cells, transplantation will affect xenografts as well as rodent tumor lines. However, our previous analyses (Fig. 5) comparing the hypoxic fractions of autochthenous mouse mammary tumors, early transplant passages of mouse mammary tumors, long-passaged mouse mammary tumor lines, and cell culture-adapted mouse mammary tumor lines ( 16) provided no evidence that the transplantation history of rodent tumors influenced their oxygenation. Thus, transplantation per se does not appear to influence tumor oxygenation. The similarity of the hypoxic fractions of human tumor xenografts and transplanted rodent tumors eliminates some possible bases for postulating a systematic difference between the hypoxic fractions of human cancers and those of the transplanted rodent tumors often used in radiobiology studies. These studies provide an additional reason to expect that studies using transplanted rodent tumors to study tumor oxygenation, reoxygenation, and the modulation of tumor oxygenation during radiotherapy can be extrapolated to predict and interpret the situation in human tumors.
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21. Rofstad, E. K.; Brustad. T. The radiosensitizing effect of metronidazole and misonidazole (Ro-07-0582) on a human malignant melanoma grown in the athymic nude mouse. Br. J. Radiol. 5 I:38 I-386; 1978. 22. Rofstad, E. K.; Fenton, B. M.; Sutherland, R. M. Intracapillary HbOz saturations in murine tumours and human tumour xenografts measured by cryospectrophotometry: relationship to tumour volume, tumour pH and fraction of radiobiologically hypoxic cells. Br. J. Cancer 57:494-502; 1988. 23. Steel, G. G.; Peckham, M. J. Human tumour xenografts: a critical appraisal. Br. J. Cancer (Suppl. IV): 133- 14 1; 1980.
July 1990, Volume 19. Number 1 24. Suit, H. D.; Zeitman, A.; Tomkinson, K.; Ramsay, J.; Gerweek. L.; Sedlacek. R. 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. 25. Urtasun, R. C.; Chapman, J. D.; Raleigh, J. A.; Franko, A. J.; Koch, C. J. Binding of ‘H-misonidazole to solid human tumors as a measure of tumor hypoxia. Int. J. Radiat. Oncol. Biol. Phys. 12: 1263- 1267: 1986. 26. Zar. J. H. Biostatistical analysis, 2nd edition. Cliffs, N.J.: Prentice-Hall. Inc.: 1984.
Englewood
