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Differentiation between hypoxic and non-hypoxic experimental tumors by dynamic contrast-enhanced magnetic resonance imaging
Radiotherapy and Oncology 98 (2011) 360–364
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MRI hypoxic imaging
Differentiation between hypoxic and non-hypoxic experimental tumors by dynamic contrast-enhanced magnetic resonance imaging Kristine Gulliksrud, Kirsti Marie Øvrebø, Berit Mathiesen, Einar K. Rofstad ⇑ Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Norway
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Article history: Received 23 October 2010 Received in revised form 15 December 2010 Accepted 23 December 2010 Available online 22 January 2011 Keywords: Dynamic contrast-enhanced MRI Hypoxia Melanoma xenografts
a b s t r a c t Background and purpose: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been suggested to be a useful method for detecting tumor hypoxia. In this study, we investigated whether DCE-MRI can differentiate between hypoxic and non-hypoxic experimental tumors. Materials and methods: Three tumor models with hypoxic tissue and three tumor models without hypoxic tissue were subjected to DCE-MRI. Parametric images of Ktrans (the volume transfer constant of Gd-DTPA) and ve (the fractional distribution volume of Gd-DTPA) were produced by pharmacokinetic analysis of the DCE-MRI series. Tumor oxygenation status was assessed by using a radiobiological assay and a pimonidazole-based immunohistochemical assay. Tumor response to fractionated irradiation (six fractions of 2 Gy in 60 h) was measured in vitro by using a clonogenic assay. Results: Tumors with hypoxic regions were more resistant to radiation treatment than were tumors without hypoxia. Ktrans was significantly higher for radiation sensitive tumors without hypoxia than for radiation resistant tumors with hypoxic regions, whereas ve did not differ significantly between non-hypoxic and hypoxic tumors. Conclusion: This study supports the clinical attempts to establish DCE-MRI as a noninvasive method for providing useful biomarkers for personalized radiation therapy. Ó 2011 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 98 (2011) 360–364
Regions with hypoxic tissue (pO2 < 10 mm Hg) are a characteristic feature of many tumors [1]. Tumor hypoxia may cause resistance to treatment and promote metastatic spread [2]. Studies of several histological types of cancer have suggested that patients with hypoxic tumors may benefit from particularly aggressive treatment [3]. A noninvasive method for detecting tumors with significant hypoxia is therefore highly required [4–6]. Preliminary preclinical and clinical studies have suggested that valid information on the oxygen tension in tumors may be obtained by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) [7,8]. The potential of gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA)-based DCE-MRI in detecting hypoxic regions in tumors is currently being evaluated in our laboratory by using xenografted human tumors as preclinical models of human cancer [9–13]. In these studies, parametric images of Ktrans and ve are produced by subjecting DCE-MRI series to pharmacokinetic analysis using the modified Kety model developed by Tofts et al. [14]. The studies carried out thus far have shown that our experimental procedure produces highly reproducible parametric images [9,10] and that the parametric images may provide valuable information on ⇑ Corresponding author. Address: Group of Radiation Biology and Tumor Physiology, Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Box 4953 Nydalen, 0424 Oslo, Norway. E-mail address: [email protected] (E.K. Rofstad). 0167-8140/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.radonc.2010.12.016
blood perfusion, extracellular volume fraction, and the extent of hypoxia in tumors [11–13]. In the present study, the potential of DCE-MRI in providing information on the oxygenation status of tumors was investigated further by subjecting tumors with hypoxic tissue and tumors without hypoxic tissue to DCE-MRI. Tumors of two human melanoma xenograft lines and two transplantation sites were included in the study. The study was based on earlier investigations in several laboratories having shown that the microvascular network and the extent of hypoxia in experimental tumors are influenced significantly by the angiogenic profile of the tumor cells and the vascularity of the transplantation site [12,15–17]. The main purpose of the study was to investigate whether DCE-MRI has the potential to distinguish tumors with hypoxic tissue from tumors without hypoxia. Materials and methods Tumor models A-07 and R-18 human melanoma xenografts growing in adult female BALB/c nu/nu mice were used as tumor models [18]. Tumors were initiated from cells cultured in RPMI-1640 medium supplemented with 13% bovine calf serum, 250 mg/l penicillin, and 50 mg/l streptomycin. Approximately 3.5 105 cells in 10 ll of Hanks’ balanced salt solution (HBSS) were inoculated intrader-
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mally (i.d.) or intramuscularly (i.m.) in the leg. Tumors with volumes of 75–150 mm3 (small tumors) or 300–600 mm3 (large tumors) were included in experiments. Three tumor models without hypoxia (small A-07 i.d., small A-07 i.m., and small R-18 i.d.) and three tumor models with hypoxic tissue (large A-07 i.d., large A-07 i.m., and large R-18 i.d.) were studied. Tumor irradiation and DCE-MRI were carried out with mice anesthetized with fentanyl citrate (Janssen Pharmaceutica, Beerse, Belgium), fluanisone (Janssen Pharmaceutica), and midazolam (Hoffmann-La Roche, Basel, Switzerland) in doses of 0.63, 20, and 10 mg/kg, respectively. Animal care and experimental procedures were in accordance with the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Marketing, and Education (New York Academy of Sciences, New York, NY).
Tumor irradiation and cell survival Tumors were irradiated under air-breathing or hypoxic conditions at a dose rate of 5.1 Gy/min, using an X-ray unit operated at 220 kV, 19–20 mA, and with 0.5-mm Cu filtration. Hypoxic tumors were obtained by euthanizing the host mice 5 min before the radiation exposure. Cell surviving fractions were measured in vitro. The tumors were given single dose irradiation or fractionated radiation treatment (six fractions of 2 Gy in 60 h). They were resected immediately after the radiation exposure, minced in cold HBSS, and treated with an enzyme solution (0.2% collagenase, 0.05% Pronase, and 0.02% DNase) at 37 °C for 2 h. Tumor volume was measured prior to the irradiation, and the cell yield was determined as the total number of trypan blue–negative cells divided by the tumor volume. Trypan blue–negative cells were plated in 25cm2 tissue culture flasks and incubated at 37 °C for 14 days for colony formation [19]. Cell surviving fractions were calculated from the cell yield, the number of cells seeded, and the number of colonies counted, corrected for the mean cell yield and the mean plating efficiency of the cells of six untreated control tumors, i.e., the cell surviving fractions of tumors given fractionated radiation treatment were measured relative to the number of clonogenic cells in the tumors before the first radiation exposure [20].
DCE-MRI DCE-MRI was carried out as described earlier [13]. Briefly, Gd-DTPA (Schering, Berlin, Germany) was administered in a bolus dose of 0.3 mmol/kg. T1-weighted images (TR = 200 ms, TE = 3.2 ms, and aT1 = 80°) were recorded at a spatial resolution of 0.31 0.31 2.0 mm3 and a time resolution of 14 s by using a 1.5-T whole-body scanner (Signa; General Electric, Milwaukee, WI) and a slotted tube resonator transceiver coil constructed for mice. Two calibration tubes, one with 0.5 mmol/l Gd-DTPA in 0.9% saline and the other with 0.9% saline only, were placed adjacent to the mice in the coil. The tumors were imaged axially in a single section through the tumor center by using an image matrix of 256 128, a field of view of 8 4 cm2, and one excitation. Two proton density images (TR = 900 ms, TE = 3.2 ms, and aPD = 20°) and three T1-weighted images were acquired before Gd-DTPA was administered, and T1-weighted images were recorded for 15 min after the administration of Gd-DTPA. Gd-DTPA concentrations were calculated from signal intensities by using the method of Hittmair et al. [21]. The DCE-MRI series were analyzed on a voxel-by-voxel basis by using the arterial input function of Benjaminsen et al. [9] and the Tofts pharmacokinetic model [14]. Parametric images of Ktrans (the volume transfer constant of Gd-DTPA) and ve (the fractional distribution volume of Gd-DTPA) were generated by using the SigmaPlot software (SPSS Science, Chicago, IL, USA). Median values of Ktrans and ve were calculated for the viable tissue of
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the tumors by excluding the voxels in necroses, using a procedure described elsewhere [15]. Histological examinations Pimonidazole [1-[(2-hydroxy-3-piperidinyl)-propyl]-2-nitroimidazole] was used as a marker of tumor hypoxia [22]. The tumors were fixed in phosphate-buffered 4% paraformaldehyde immediately after the DCE-MRI, and histological sections were prepared by using standard procedures. Immunohistochemistry was performed by using an avidin–biotin peroxidase-based staining method [19]. An anti-pimonidazole rabbit polyclonal antibody (gift from Professor Raleigh, Department of Radiation Oncology, University of North Carolina School of Medicine, Chapel Hill, NC) was used as primary antibody. Diaminobenzidine was used as chromogen, and hematoxylin was used for counterstaining. Three crosssections were examined for each tumor. Statistical analysis Experimental data are presented as arithmetic mean ± SE unless otherwise stated. Statistical analyses were carried out by one-way ANOVA followed by the Student Newman–Keuls test. The method of Kolmogorov–Smirnov was used to test for normality. Probability values of P < 0.05 were considered significant. The statistical analysis was carried out by using the SigmaStat statistical software (SPSS Science). Results Tumors of all six models were exposed to single graded radiation doses under air-breathing or hypoxic conditions to assess the radiation sensitivity of the tumor cells and the oxygenation status of the tumors. The D0 values of the cell survival curves were found to be 0.89 ± 0.08 Gy (air-breathing conditions) and 2.51 ± 0.10 Gy (hypoxic conditions) for small A-07 i.d. tumors, 2.52 ± 0.11 Gy (air-breathing conditions) and 2.56 ± 0.13 Gy (hypoxic conditions) for large A-07 i.d. tumors, 0.88 ± 0.09 Gy (airbreathing conditions) and 2.54 ± 0.12 Gy (hypoxic conditions) for small A-07 i.m. tumors, 2.49 ± 0.13 Gy (air-breathing conditions) and 2.53 ± 0.12 Gy (hypoxic conditions) for large A-07 i.m. tumors, 1.05 ± 0.10 Gy (air-breathing conditions) and 2.99 ± 0.11 Gy (hypoxic conditions) for small R-18 i.d. tumors, and 3.02 ± 0.10 Gy (air-breathing conditions) and 2.97 ± 0.12 Gy (hypoxic conditions) for large R-18 i.d. tumors (Fig. 1). The survival curves of the tumors irradiated under air-breathing conditions were either steeper than (small tumors) or parallel to (large tumors) those of the tumors irradiated under hypoxic conditions. The oxygen enhancement ratios of the small tumors were calculated from the slopes of the survival curves to be 2.82 ± 0.28 (A-07 i.d.), 2.89 ± 0.33 (A-07 i.m.), and 2.85 ± 0.29 (R-18 i.d.). The survival curves of the small tumors were thus consistent with no or insignificant hypoxia in the tumors. The fractions of hypoxic cells of the large tumors were calculated from the vertical displacement of the survival curves to be 8 ± 3% (A-07 i.d.), 25 ± 5% (A-07 i.m.), and 58 ± 10% (R-18 i.d.). Response to fractionated irradiation was studied by irradiating tumors in air-breathing mice with six fractions of 2 Gy in 60 h. The fraction of surviving cells was significantly higher for the three tumor models with hypoxic cells than for the three tumor models without hypoxia (Fig. 2; P < 0.001). Furthermore, the surviving fraction was significantly higher for large R-18 i.d. tumors than for large A-07 i.m. tumors (P < 0.01) and significantly higher for large A-07 i.m. tumors than for large A-07 i.d. tumors (P < 0.05), i.e., it decreased with decreasing fraction of hypoxic cells. Tumors of all models were subjected to DCE-MRI to investigate whether Ktrans and ve images can differentiate between tumors
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Fig. 1. Cell survival curves for small A-07 i.d. tumors, large A-07 i.d. tumors, small A-07 i.m. tumors, large A-07 i.m. tumors, small R-18 i.d. tumors, and large R-18 i.d. tumors treated with single radiation doses under air-breathing (s) or hypoxic (d) conditions. Points and bars represent the geometric mean ± SE of 5–7 tumors. The curves were fitted to the data by regression analysis.
Fig. 3. The Ktrans image, Ktrans frequency distribution, ve image, and ve frequency distribution of a representative small A-07 i.d. tumor (a) and a representative large A-07 i.d. tumor (b), and immunohistochemical preparations of these tumors showing positive pimonidazole staining in the large tumor, but not in the small tumor (c).
Fig. 2. Cell surviving fractions for small A-07 i.d. tumors, large A-07 i.d. tumors, small A-07 i.m. tumors, large A-07 i.m. tumors, small R-18 i.d. tumors, and large R18 i.d. tumors given fractionated radiation treatment (six fractions of 2 Gy in 60 h) under air-breathing conditions. Columns and bars represent the geometric mean ± SE of 5–7 tumors.
without hypoxia and tumors with significant fractions of hypoxic cells. Representative DCE-MRI data are presented in Fig. 3, which shows the Ktrans image, the Ktrans frequency distribution, the ve image, and the ve frequency distribution of a small (Fig. 3a) and a large (Fig. 3b) A-07 i.d. tumor. The tumors were highly heterogeneous in Ktrans with the highest values in the periphery and the lowest values in central regions. The intratumor heterogeneity in ve was also significant, particularly in the large tumors.
Histological examinations of the imaged tumors showed that most of the large A-07 i.m. tumors had developed regions with necrotic tissue, whereas significant necrosis could not be detected in any of the other tumors. Furthermore, all large tumors showed regions with positive pimonidazole staining, whereas none of the small tumors stained positive for pimonidazole, as exemplified in Fig. 3c. The immunohistochemical observations were thus consistent with the radiobiological data. Median Ktrans was significantly higher for the three tumor models without hypoxia than for the three tumor models with hypoxic cells [Fig. 4a; P < 0.001 (A-07 i.d. and A-07 i.m.) and P < 0.05 (R-18 i.d.)]. There was hardly any overlap between the median Ktrans values of the hypoxic and the non-hypoxic tumors. Thus, the highest value measured for any large A-07 i.d. or i.m. tumor was lower than the lowest value measured for any of the small tumors, and only one small A-07 i.m. tumor and one small R-18 i.d. tumor
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Fig. 4. Median Ktrans (a) and median ve (b) of small A-07 i.d. tumors, large A-07 i.d. tumors, small A-07 i.m. tumors, large A-07 i.m. tumors, small R-18 i.d. tumors, and large R-18 i.d. tumors. Points and horizontal bars represent individual tumors and mean values, respectively.
showed a lower value than the highest value measured for any large R-18 i.d. tumor. On the other hand, median ve did not differ significantly between small and large A-07 i.d. tumors, between small and large A-07 i.m. tumors, or between small and large R18 i.d. tumors (Fig. 4b; P > 0.05). Even though large A-07 i.d. tumors, large A-07 i.m. tumors, and large R-18 i.d. tumors differed significantly in fraction of hypoxic cells and response to fractionated irradiation, they did not differ significantly in median Ktrans (Fig. 4a; P > 0.05). However, median ve was significantly higher for large A-07 tumors growing i.d. or i.m. than for large R-18 i.d. tumors (Fig. 4b; P < 0.001). Discussion The possibility that DCE-MRI may be a useful noninvasive method for differentiating between hypoxic and non-hypoxic tumors was investigated by using three tumor models with hypoxic regions and three tumor models without hypoxic tissue. Radiobiological experiments showed that small A-07 i.d. tumors, small A-07 i.m. tumors, and small R-18 i.d. tumors did not possess hypoxic cells, whereas the fraction of hypoxic cells was significant in large A-07 i.d. tumors, large A-07 i.m. tumors, and large R-18 i.d. tumors. These findings were verified by examining histological sections immunostained for the hypoxia marker pimonidazole, and were consistent with previous studies having shown that the TCD50 is 16.8–18.9 Gy for 100-mm3 A-07 i.d. tumors, 20.5–22.6 Gy for 100-mm3 R-18 i.d. tumors, and 31.7–37.7 Gy for 300-mm3 A07 i.d. tumors [23,24]. Consequently, our tumor models should be suitable for providing valid information on the primary question addressed in the present study. The development of hypoxia in tumors depends on several factors including the vascularity of the surrounding normal tissue, the potential of the tumor cells to synthesize and secrete angiogenic factors, and the respiratory activity of the tumor cells and, hence, the cell density of the tumor tissue [25–27]. The tumor models included in the present study differed significantly in these proper-
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ties. Compared with most experimental tumors, the cell density is low in A-07 tumors and high in R-18 tumors [12,13]. The rate of secretion of vascular endothelial growth factor-A and interleukin-8 is higher in A-07 cells than in R-18 cells by factors of 50 and 150, respectively, and in contrast to the R-18 cells, the A07 cells show significant expression of platelet-derived endothelial cell growth factor and basic fibroblast growth factor [28]. Because skin and muscle tissue show highly different vascularity [29], both A-07 i.d. tumors and A-07 i.m. tumors were studied. R-18 i.m. tumors were not included in the study, however, because preliminary experiments revealed that many small R-18 i.m tumors stain positive for pimonidazole and show significant fractions of radiobiologically hypoxic cells. The response to fractionated radiation treatment was associated with the oxygenation status of the tumor models. Thus, the cell survival after exposure to six fractions of 2 Gy was higher for the models with hypoxic tissue than for the models without hypoxia, and it decreased with decreasing fraction of hypoxic cells. This observation suggests that any reoxygenation between the fractions was inadequate and that the radiation response was limited by the hypoxic cells of the tumors. The tumor models thus showed a response to fractionated radiation treatment that was consistent with the clinical observations that tumor hypoxia may cause resistance to radiation therapy [3,30,31]. It should be noticed, however that a treatment consisting of six fractions of 2 Gy constitutes only approximately one fifth of a complete clinically relevant treatment regimen. Others have measured the oxygenation of experimental tumors noninvasively and shown that the response to radiation treatment may be associated with tumor oxygenation status. For example, studies of sublines of the Dunning R3327 rat prostate carcinoma showed that tissue pO2 was higher in small tumors than in their larger counterparts and that tumor growth delay after single dose irradiation with 30 Gy increased with increasing pO2 as assessed by using 19F nuclear magnetic resonance echo planar imaging relaxometry of the reporter molecule hexafluorobenzene [32,33]. Our DCE-MRI recordings were analyzed by using the modified Kety pharmacokinetic model [14]. We have reported previously that this model gives good fits to our data [34] and that Monte Carlo analysis has revealed that the signal-to-noise ratio is sufficiently high that Ktrans and ve images are not influenced significantly by noise [35], methodological conditions that were verified to be valid also in the present study. Importantly, by subjecting the same tumors to DCE-MRI twice with an interimaging interval of 60 min, we have shown that highly reproducible Ktrans and ve images are produced by the experimental procedure used herein [9,10]. The strengths and weaknesses of our DCE-MRI method have been reviewed thoroughly elsewhere [12,13,34,35]. The Ktrans images mirrored the oxygenation status and the radiation response of the tumors well. Thus, median Ktrans was significantly higher for the radiation sensitive tumor models without hypoxic tissue than for the radiation resistant models with hypoxic tissue. In fact, there was little overlap between median Ktrans for the hypoxic, radiation resistant and the non-hypoxic, radiation sensitive tumors, regardless of tumor model. It should be noticed, however, that the cell surviving fraction after six fractions of 2 Gy was 18-fold higher for the large than for the small R-18 i.d. tumors, whereas mean Ktrans for the two tumor groups differed by a factor of only 1.8. The present DCE-MRI method may thus not be sufficiently sensitive to discriminate well between the radiation response of non-hypoxic tumors and tumors with very low hypoxic fractions. The ve images did not differ significantly between the tumor models with hypoxic tissue and the corresponding models without hypoxia and thus did not reflect the oxygenation status or the radiation response of the tumors. Taken together, our data suggest that
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the potential of Ktrans images in differentiating between radiation resistant tumors with significant hypoxia and radiation sensitive tumors without hypoxia may be significant, whereas ve images may not be helpful for this purpose. It is noteworthy, however, that the DCE-MRI and radiation treatment experiments reported herein were not carried out on the same individual tumors, but on different cohorts of similar tumors. Median Ktrans did not differ significantly between large A-07 i.d. tumors, large A-07 i.m. tumors, and large R-18 i.d. tumors even though the mean fraction of hypoxic cells differed substantially among these tumor models, from 8% in the large A-07 i.d. tumors to 58% in the large R-18 i.d. tumors. This observation is consistent with the main conclusions of two previous DCE-MRI studies carried out in our laboratory [12,15]. First, the extent of hypoxia in experimental tumors cannot be assessed quantitatively by DCE-MRI without taking Ktrans as well as ve images into consideration [12]. Second, quantitative assessment of hypoxia in experimental tumors by DCE-MRI may require the use of tumor site-specific criteria when translating parametric MR images into hypoxic fractions [15]. The latter study involved A-07 i.d. and i.m. tumors with volumes of 200–600 mm3, and it showed that the i.d. and i.m. tumors did not differ significantly in E F [E F is tumor blood perfusion in units of ml/(g min) and is related to Ktrans by the following expression: E F = Ktrans/q (1 Hct), where q is the density of the tumor tissue in units of g/ml, and Hct is the hematocrit] although they differed significantly in fraction of hypoxic cells, microvascular density, and vessel diameter distribution [15]. Clinical studies have suggested that DCE-MRI may provide useful prognostic information on cancer patients, and have increased the interest in using DCE-MRI to characterize the oxygenation status of tumors given radiation therapy [36]. The present preclinical study supports the clinical attempts to establish Gd-DTPA-based DCE-MRI as a noninvasive method for differentiating between radiation resistant hypoxic tumors and radiation sensitive non-hypoxic tumors. The potential of DCE-MRI in providing useful biomarkers for personalized radiation therapy merits increased clinical interest. Acknowledgments Financial support was received from the Norwegian Cancer Society and the South-Eastern Norway Regional Health Authority. References [1] Höckel M, Vaupel P. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst 2001;93:266–76. [2] Wouters BG, Weppler SA, Koritzinsky M, et al. Hypoxia as a target for combined modality treatments. Eur J Cancer 2002;38:240–57. [3] Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev 2007;26:225–39. [4] Tatum JL, Kelloff GJ, Gillies RJ, et al. Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy. Int J Radiat Biol 2006;82:699–757. [5] Troost EGC, Schinagl DAX, Bussink J, Oyen WJG, Kaanders JHAM. Clinical evidence on PET-CT for radiation therapy planning in head and neck tumours. Radiother Oncol 2010;96:328–34. [6] Thorwarth D, Alber M. Implementation of hypoxia imaging into treatment planning and delivery. Radiother Oncol 2010;97:172–5. [7] Wang Z, Su MY, Nalcioglu O. Applications of dynamic contrast enhanced MRI in oncology: measurement of tumor oxygen tension. Technol Cancer Res Treat 2002;1:29–38. [8] Cooper RA, Carrington BM, Loncaster JA, et al. Tumour oxygenation levels correlate with dynamic contrast-enhanced magnetic resonance imaging parameters in carcinoma of the cervix. Radiother Oncol 2000;57:53–9. [9] Benjaminsen IC, Graff BA, Brurberg KG, Rofstad EK. Assessment of tumor blood perfusion by high-resolution dynamic contrast-enhanced MRI: a preclinical study of human melanoma xenografts. Magn Reson Med 2004;52:269–76.
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MRI hypoxic imaging
Differentiation between hypoxic and non-hypoxic experimental tumors by dynamic contrast-enhanced magnetic resonance imaging Kristine Gulliksrud, Kirsti Marie Øvrebø, Berit Mathiesen, Einar K. Rofstad ⇑ Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Norway
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Article history: Received 23 October 2010 Received in revised form 15 December 2010 Accepted 23 December 2010 Available online 22 January 2011 Keywords: Dynamic contrast-enhanced MRI Hypoxia Melanoma xenografts
a b s t r a c t Background and purpose: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been suggested to be a useful method for detecting tumor hypoxia. In this study, we investigated whether DCE-MRI can differentiate between hypoxic and non-hypoxic experimental tumors. Materials and methods: Three tumor models with hypoxic tissue and three tumor models without hypoxic tissue were subjected to DCE-MRI. Parametric images of Ktrans (the volume transfer constant of Gd-DTPA) and ve (the fractional distribution volume of Gd-DTPA) were produced by pharmacokinetic analysis of the DCE-MRI series. Tumor oxygenation status was assessed by using a radiobiological assay and a pimonidazole-based immunohistochemical assay. Tumor response to fractionated irradiation (six fractions of 2 Gy in 60 h) was measured in vitro by using a clonogenic assay. Results: Tumors with hypoxic regions were more resistant to radiation treatment than were tumors without hypoxia. Ktrans was significantly higher for radiation sensitive tumors without hypoxia than for radiation resistant tumors with hypoxic regions, whereas ve did not differ significantly between non-hypoxic and hypoxic tumors. Conclusion: This study supports the clinical attempts to establish DCE-MRI as a noninvasive method for providing useful biomarkers for personalized radiation therapy. Ó 2011 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 98 (2011) 360–364
Regions with hypoxic tissue (pO2 < 10 mm Hg) are a characteristic feature of many tumors [1]. Tumor hypoxia may cause resistance to treatment and promote metastatic spread [2]. Studies of several histological types of cancer have suggested that patients with hypoxic tumors may benefit from particularly aggressive treatment [3]. A noninvasive method for detecting tumors with significant hypoxia is therefore highly required [4–6]. Preliminary preclinical and clinical studies have suggested that valid information on the oxygen tension in tumors may be obtained by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) [7,8]. The potential of gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA)-based DCE-MRI in detecting hypoxic regions in tumors is currently being evaluated in our laboratory by using xenografted human tumors as preclinical models of human cancer [9–13]. In these studies, parametric images of Ktrans and ve are produced by subjecting DCE-MRI series to pharmacokinetic analysis using the modified Kety model developed by Tofts et al. [14]. The studies carried out thus far have shown that our experimental procedure produces highly reproducible parametric images [9,10] and that the parametric images may provide valuable information on ⇑ Corresponding author. Address: Group of Radiation Biology and Tumor Physiology, Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Box 4953 Nydalen, 0424 Oslo, Norway. E-mail address: [email protected] (E.K. Rofstad). 0167-8140/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.radonc.2010.12.016
blood perfusion, extracellular volume fraction, and the extent of hypoxia in tumors [11–13]. In the present study, the potential of DCE-MRI in providing information on the oxygenation status of tumors was investigated further by subjecting tumors with hypoxic tissue and tumors without hypoxic tissue to DCE-MRI. Tumors of two human melanoma xenograft lines and two transplantation sites were included in the study. The study was based on earlier investigations in several laboratories having shown that the microvascular network and the extent of hypoxia in experimental tumors are influenced significantly by the angiogenic profile of the tumor cells and the vascularity of the transplantation site [12,15–17]. The main purpose of the study was to investigate whether DCE-MRI has the potential to distinguish tumors with hypoxic tissue from tumors without hypoxia. Materials and methods Tumor models A-07 and R-18 human melanoma xenografts growing in adult female BALB/c nu/nu mice were used as tumor models [18]. Tumors were initiated from cells cultured in RPMI-1640 medium supplemented with 13% bovine calf serum, 250 mg/l penicillin, and 50 mg/l streptomycin. Approximately 3.5 105 cells in 10 ll of Hanks’ balanced salt solution (HBSS) were inoculated intrader-
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mally (i.d.) or intramuscularly (i.m.) in the leg. Tumors with volumes of 75–150 mm3 (small tumors) or 300–600 mm3 (large tumors) were included in experiments. Three tumor models without hypoxia (small A-07 i.d., small A-07 i.m., and small R-18 i.d.) and three tumor models with hypoxic tissue (large A-07 i.d., large A-07 i.m., and large R-18 i.d.) were studied. Tumor irradiation and DCE-MRI were carried out with mice anesthetized with fentanyl citrate (Janssen Pharmaceutica, Beerse, Belgium), fluanisone (Janssen Pharmaceutica), and midazolam (Hoffmann-La Roche, Basel, Switzerland) in doses of 0.63, 20, and 10 mg/kg, respectively. Animal care and experimental procedures were in accordance with the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Marketing, and Education (New York Academy of Sciences, New York, NY).
Tumor irradiation and cell survival Tumors were irradiated under air-breathing or hypoxic conditions at a dose rate of 5.1 Gy/min, using an X-ray unit operated at 220 kV, 19–20 mA, and with 0.5-mm Cu filtration. Hypoxic tumors were obtained by euthanizing the host mice 5 min before the radiation exposure. Cell surviving fractions were measured in vitro. The tumors were given single dose irradiation or fractionated radiation treatment (six fractions of 2 Gy in 60 h). They were resected immediately after the radiation exposure, minced in cold HBSS, and treated with an enzyme solution (0.2% collagenase, 0.05% Pronase, and 0.02% DNase) at 37 °C for 2 h. Tumor volume was measured prior to the irradiation, and the cell yield was determined as the total number of trypan blue–negative cells divided by the tumor volume. Trypan blue–negative cells were plated in 25cm2 tissue culture flasks and incubated at 37 °C for 14 days for colony formation [19]. Cell surviving fractions were calculated from the cell yield, the number of cells seeded, and the number of colonies counted, corrected for the mean cell yield and the mean plating efficiency of the cells of six untreated control tumors, i.e., the cell surviving fractions of tumors given fractionated radiation treatment were measured relative to the number of clonogenic cells in the tumors before the first radiation exposure [20].
DCE-MRI DCE-MRI was carried out as described earlier [13]. Briefly, Gd-DTPA (Schering, Berlin, Germany) was administered in a bolus dose of 0.3 mmol/kg. T1-weighted images (TR = 200 ms, TE = 3.2 ms, and aT1 = 80°) were recorded at a spatial resolution of 0.31 0.31 2.0 mm3 and a time resolution of 14 s by using a 1.5-T whole-body scanner (Signa; General Electric, Milwaukee, WI) and a slotted tube resonator transceiver coil constructed for mice. Two calibration tubes, one with 0.5 mmol/l Gd-DTPA in 0.9% saline and the other with 0.9% saline only, were placed adjacent to the mice in the coil. The tumors were imaged axially in a single section through the tumor center by using an image matrix of 256 128, a field of view of 8 4 cm2, and one excitation. Two proton density images (TR = 900 ms, TE = 3.2 ms, and aPD = 20°) and three T1-weighted images were acquired before Gd-DTPA was administered, and T1-weighted images were recorded for 15 min after the administration of Gd-DTPA. Gd-DTPA concentrations were calculated from signal intensities by using the method of Hittmair et al. [21]. The DCE-MRI series were analyzed on a voxel-by-voxel basis by using the arterial input function of Benjaminsen et al. [9] and the Tofts pharmacokinetic model [14]. Parametric images of Ktrans (the volume transfer constant of Gd-DTPA) and ve (the fractional distribution volume of Gd-DTPA) were generated by using the SigmaPlot software (SPSS Science, Chicago, IL, USA). Median values of Ktrans and ve were calculated for the viable tissue of
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the tumors by excluding the voxels in necroses, using a procedure described elsewhere [15]. Histological examinations Pimonidazole [1-[(2-hydroxy-3-piperidinyl)-propyl]-2-nitroimidazole] was used as a marker of tumor hypoxia [22]. The tumors were fixed in phosphate-buffered 4% paraformaldehyde immediately after the DCE-MRI, and histological sections were prepared by using standard procedures. Immunohistochemistry was performed by using an avidin–biotin peroxidase-based staining method [19]. An anti-pimonidazole rabbit polyclonal antibody (gift from Professor Raleigh, Department of Radiation Oncology, University of North Carolina School of Medicine, Chapel Hill, NC) was used as primary antibody. Diaminobenzidine was used as chromogen, and hematoxylin was used for counterstaining. Three crosssections were examined for each tumor. Statistical analysis Experimental data are presented as arithmetic mean ± SE unless otherwise stated. Statistical analyses were carried out by one-way ANOVA followed by the Student Newman–Keuls test. The method of Kolmogorov–Smirnov was used to test for normality. Probability values of P < 0.05 were considered significant. The statistical analysis was carried out by using the SigmaStat statistical software (SPSS Science). Results Tumors of all six models were exposed to single graded radiation doses under air-breathing or hypoxic conditions to assess the radiation sensitivity of the tumor cells and the oxygenation status of the tumors. The D0 values of the cell survival curves were found to be 0.89 ± 0.08 Gy (air-breathing conditions) and 2.51 ± 0.10 Gy (hypoxic conditions) for small A-07 i.d. tumors, 2.52 ± 0.11 Gy (air-breathing conditions) and 2.56 ± 0.13 Gy (hypoxic conditions) for large A-07 i.d. tumors, 0.88 ± 0.09 Gy (airbreathing conditions) and 2.54 ± 0.12 Gy (hypoxic conditions) for small A-07 i.m. tumors, 2.49 ± 0.13 Gy (air-breathing conditions) and 2.53 ± 0.12 Gy (hypoxic conditions) for large A-07 i.m. tumors, 1.05 ± 0.10 Gy (air-breathing conditions) and 2.99 ± 0.11 Gy (hypoxic conditions) for small R-18 i.d. tumors, and 3.02 ± 0.10 Gy (air-breathing conditions) and 2.97 ± 0.12 Gy (hypoxic conditions) for large R-18 i.d. tumors (Fig. 1). The survival curves of the tumors irradiated under air-breathing conditions were either steeper than (small tumors) or parallel to (large tumors) those of the tumors irradiated under hypoxic conditions. The oxygen enhancement ratios of the small tumors were calculated from the slopes of the survival curves to be 2.82 ± 0.28 (A-07 i.d.), 2.89 ± 0.33 (A-07 i.m.), and 2.85 ± 0.29 (R-18 i.d.). The survival curves of the small tumors were thus consistent with no or insignificant hypoxia in the tumors. The fractions of hypoxic cells of the large tumors were calculated from the vertical displacement of the survival curves to be 8 ± 3% (A-07 i.d.), 25 ± 5% (A-07 i.m.), and 58 ± 10% (R-18 i.d.). Response to fractionated irradiation was studied by irradiating tumors in air-breathing mice with six fractions of 2 Gy in 60 h. The fraction of surviving cells was significantly higher for the three tumor models with hypoxic cells than for the three tumor models without hypoxia (Fig. 2; P < 0.001). Furthermore, the surviving fraction was significantly higher for large R-18 i.d. tumors than for large A-07 i.m. tumors (P < 0.01) and significantly higher for large A-07 i.m. tumors than for large A-07 i.d. tumors (P < 0.05), i.e., it decreased with decreasing fraction of hypoxic cells. Tumors of all models were subjected to DCE-MRI to investigate whether Ktrans and ve images can differentiate between tumors
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Fig. 1. Cell survival curves for small A-07 i.d. tumors, large A-07 i.d. tumors, small A-07 i.m. tumors, large A-07 i.m. tumors, small R-18 i.d. tumors, and large R-18 i.d. tumors treated with single radiation doses under air-breathing (s) or hypoxic (d) conditions. Points and bars represent the geometric mean ± SE of 5–7 tumors. The curves were fitted to the data by regression analysis.
Fig. 3. The Ktrans image, Ktrans frequency distribution, ve image, and ve frequency distribution of a representative small A-07 i.d. tumor (a) and a representative large A-07 i.d. tumor (b), and immunohistochemical preparations of these tumors showing positive pimonidazole staining in the large tumor, but not in the small tumor (c).
Fig. 2. Cell surviving fractions for small A-07 i.d. tumors, large A-07 i.d. tumors, small A-07 i.m. tumors, large A-07 i.m. tumors, small R-18 i.d. tumors, and large R18 i.d. tumors given fractionated radiation treatment (six fractions of 2 Gy in 60 h) under air-breathing conditions. Columns and bars represent the geometric mean ± SE of 5–7 tumors.
without hypoxia and tumors with significant fractions of hypoxic cells. Representative DCE-MRI data are presented in Fig. 3, which shows the Ktrans image, the Ktrans frequency distribution, the ve image, and the ve frequency distribution of a small (Fig. 3a) and a large (Fig. 3b) A-07 i.d. tumor. The tumors were highly heterogeneous in Ktrans with the highest values in the periphery and the lowest values in central regions. The intratumor heterogeneity in ve was also significant, particularly in the large tumors.
Histological examinations of the imaged tumors showed that most of the large A-07 i.m. tumors had developed regions with necrotic tissue, whereas significant necrosis could not be detected in any of the other tumors. Furthermore, all large tumors showed regions with positive pimonidazole staining, whereas none of the small tumors stained positive for pimonidazole, as exemplified in Fig. 3c. The immunohistochemical observations were thus consistent with the radiobiological data. Median Ktrans was significantly higher for the three tumor models without hypoxia than for the three tumor models with hypoxic cells [Fig. 4a; P < 0.001 (A-07 i.d. and A-07 i.m.) and P < 0.05 (R-18 i.d.)]. There was hardly any overlap between the median Ktrans values of the hypoxic and the non-hypoxic tumors. Thus, the highest value measured for any large A-07 i.d. or i.m. tumor was lower than the lowest value measured for any of the small tumors, and only one small A-07 i.m. tumor and one small R-18 i.d. tumor
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Fig. 4. Median Ktrans (a) and median ve (b) of small A-07 i.d. tumors, large A-07 i.d. tumors, small A-07 i.m. tumors, large A-07 i.m. tumors, small R-18 i.d. tumors, and large R-18 i.d. tumors. Points and horizontal bars represent individual tumors and mean values, respectively.
showed a lower value than the highest value measured for any large R-18 i.d. tumor. On the other hand, median ve did not differ significantly between small and large A-07 i.d. tumors, between small and large A-07 i.m. tumors, or between small and large R18 i.d. tumors (Fig. 4b; P > 0.05). Even though large A-07 i.d. tumors, large A-07 i.m. tumors, and large R-18 i.d. tumors differed significantly in fraction of hypoxic cells and response to fractionated irradiation, they did not differ significantly in median Ktrans (Fig. 4a; P > 0.05). However, median ve was significantly higher for large A-07 tumors growing i.d. or i.m. than for large R-18 i.d. tumors (Fig. 4b; P < 0.001). Discussion The possibility that DCE-MRI may be a useful noninvasive method for differentiating between hypoxic and non-hypoxic tumors was investigated by using three tumor models with hypoxic regions and three tumor models without hypoxic tissue. Radiobiological experiments showed that small A-07 i.d. tumors, small A-07 i.m. tumors, and small R-18 i.d. tumors did not possess hypoxic cells, whereas the fraction of hypoxic cells was significant in large A-07 i.d. tumors, large A-07 i.m. tumors, and large R-18 i.d. tumors. These findings were verified by examining histological sections immunostained for the hypoxia marker pimonidazole, and were consistent with previous studies having shown that the TCD50 is 16.8–18.9 Gy for 100-mm3 A-07 i.d. tumors, 20.5–22.6 Gy for 100-mm3 R-18 i.d. tumors, and 31.7–37.7 Gy for 300-mm3 A07 i.d. tumors [23,24]. Consequently, our tumor models should be suitable for providing valid information on the primary question addressed in the present study. The development of hypoxia in tumors depends on several factors including the vascularity of the surrounding normal tissue, the potential of the tumor cells to synthesize and secrete angiogenic factors, and the respiratory activity of the tumor cells and, hence, the cell density of the tumor tissue [25–27]. The tumor models included in the present study differed significantly in these proper-
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ties. Compared with most experimental tumors, the cell density is low in A-07 tumors and high in R-18 tumors [12,13]. The rate of secretion of vascular endothelial growth factor-A and interleukin-8 is higher in A-07 cells than in R-18 cells by factors of 50 and 150, respectively, and in contrast to the R-18 cells, the A07 cells show significant expression of platelet-derived endothelial cell growth factor and basic fibroblast growth factor [28]. Because skin and muscle tissue show highly different vascularity [29], both A-07 i.d. tumors and A-07 i.m. tumors were studied. R-18 i.m. tumors were not included in the study, however, because preliminary experiments revealed that many small R-18 i.m tumors stain positive for pimonidazole and show significant fractions of radiobiologically hypoxic cells. The response to fractionated radiation treatment was associated with the oxygenation status of the tumor models. Thus, the cell survival after exposure to six fractions of 2 Gy was higher for the models with hypoxic tissue than for the models without hypoxia, and it decreased with decreasing fraction of hypoxic cells. This observation suggests that any reoxygenation between the fractions was inadequate and that the radiation response was limited by the hypoxic cells of the tumors. The tumor models thus showed a response to fractionated radiation treatment that was consistent with the clinical observations that tumor hypoxia may cause resistance to radiation therapy [3,30,31]. It should be noticed, however that a treatment consisting of six fractions of 2 Gy constitutes only approximately one fifth of a complete clinically relevant treatment regimen. Others have measured the oxygenation of experimental tumors noninvasively and shown that the response to radiation treatment may be associated with tumor oxygenation status. For example, studies of sublines of the Dunning R3327 rat prostate carcinoma showed that tissue pO2 was higher in small tumors than in their larger counterparts and that tumor growth delay after single dose irradiation with 30 Gy increased with increasing pO2 as assessed by using 19F nuclear magnetic resonance echo planar imaging relaxometry of the reporter molecule hexafluorobenzene [32,33]. Our DCE-MRI recordings were analyzed by using the modified Kety pharmacokinetic model [14]. We have reported previously that this model gives good fits to our data [34] and that Monte Carlo analysis has revealed that the signal-to-noise ratio is sufficiently high that Ktrans and ve images are not influenced significantly by noise [35], methodological conditions that were verified to be valid also in the present study. Importantly, by subjecting the same tumors to DCE-MRI twice with an interimaging interval of 60 min, we have shown that highly reproducible Ktrans and ve images are produced by the experimental procedure used herein [9,10]. The strengths and weaknesses of our DCE-MRI method have been reviewed thoroughly elsewhere [12,13,34,35]. The Ktrans images mirrored the oxygenation status and the radiation response of the tumors well. Thus, median Ktrans was significantly higher for the radiation sensitive tumor models without hypoxic tissue than for the radiation resistant models with hypoxic tissue. In fact, there was little overlap between median Ktrans for the hypoxic, radiation resistant and the non-hypoxic, radiation sensitive tumors, regardless of tumor model. It should be noticed, however, that the cell surviving fraction after six fractions of 2 Gy was 18-fold higher for the large than for the small R-18 i.d. tumors, whereas mean Ktrans for the two tumor groups differed by a factor of only 1.8. The present DCE-MRI method may thus not be sufficiently sensitive to discriminate well between the radiation response of non-hypoxic tumors and tumors with very low hypoxic fractions. The ve images did not differ significantly between the tumor models with hypoxic tissue and the corresponding models without hypoxia and thus did not reflect the oxygenation status or the radiation response of the tumors. Taken together, our data suggest that
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the potential of Ktrans images in differentiating between radiation resistant tumors with significant hypoxia and radiation sensitive tumors without hypoxia may be significant, whereas ve images may not be helpful for this purpose. It is noteworthy, however, that the DCE-MRI and radiation treatment experiments reported herein were not carried out on the same individual tumors, but on different cohorts of similar tumors. Median Ktrans did not differ significantly between large A-07 i.d. tumors, large A-07 i.m. tumors, and large R-18 i.d. tumors even though the mean fraction of hypoxic cells differed substantially among these tumor models, from 8% in the large A-07 i.d. tumors to 58% in the large R-18 i.d. tumors. This observation is consistent with the main conclusions of two previous DCE-MRI studies carried out in our laboratory [12,15]. First, the extent of hypoxia in experimental tumors cannot be assessed quantitatively by DCE-MRI without taking Ktrans as well as ve images into consideration [12]. Second, quantitative assessment of hypoxia in experimental tumors by DCE-MRI may require the use of tumor site-specific criteria when translating parametric MR images into hypoxic fractions [15]. The latter study involved A-07 i.d. and i.m. tumors with volumes of 200–600 mm3, and it showed that the i.d. and i.m. tumors did not differ significantly in E F [E F is tumor blood perfusion in units of ml/(g min) and is related to Ktrans by the following expression: E F = Ktrans/q (1 Hct), where q is the density of the tumor tissue in units of g/ml, and Hct is the hematocrit] although they differed significantly in fraction of hypoxic cells, microvascular density, and vessel diameter distribution [15]. Clinical studies have suggested that DCE-MRI may provide useful prognostic information on cancer patients, and have increased the interest in using DCE-MRI to characterize the oxygenation status of tumors given radiation therapy [36]. The present preclinical study supports the clinical attempts to establish Gd-DTPA-based DCE-MRI as a noninvasive method for differentiating between radiation resistant hypoxic tumors and radiation sensitive non-hypoxic tumors. The potential of DCE-MRI in providing useful biomarkers for personalized radiation therapy merits increased clinical interest. Acknowledgments Financial support was received from the Norwegian Cancer Society and the South-Eastern Norway Regional Health Authority. References [1] Höckel M, Vaupel P. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst 2001;93:266–76. [2] Wouters BG, Weppler SA, Koritzinsky M, et al. Hypoxia as a target for combined modality treatments. Eur J Cancer 2002;38:240–57. [3] Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev 2007;26:225–39. [4] Tatum JL, Kelloff GJ, Gillies RJ, et al. Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy. Int J Radiat Biol 2006;82:699–757. [5] Troost EGC, Schinagl DAX, Bussink J, Oyen WJG, Kaanders JHAM. Clinical evidence on PET-CT for radiation therapy planning in head and neck tumours. Radiother Oncol 2010;96:328–34. [6] Thorwarth D, Alber M. Implementation of hypoxia imaging into treatment planning and delivery. Radiother Oncol 2010;97:172–5. [7] Wang Z, Su MY, Nalcioglu O. Applications of dynamic contrast enhanced MRI in oncology: measurement of tumor oxygen tension. Technol Cancer Res Treat 2002;1:29–38. [8] Cooper RA, Carrington BM, Loncaster JA, et al. Tumour oxygenation levels correlate with dynamic contrast-enhanced magnetic resonance imaging parameters in carcinoma of the cervix. Radiother Oncol 2000;57:53–9. [9] Benjaminsen IC, Graff BA, Brurberg KG, Rofstad EK. Assessment of tumor blood perfusion by high-resolution dynamic contrast-enhanced MRI: a preclinical study of human melanoma xenografts. Magn Reson Med 2004;52:269–76.
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