British Journal of Radiology (2006) 79, 71-75
© 2006 British Institute of Radiology
doi: 10.1259/bjr/50464795
Radiosurgical palliation of aggressive murine SCCVII squamous cell carcinomas using synchrotron-generated X-ray microbeams
M Miura, PhD
1
H Blattmann, PhD
2
E Bräuer-Krisch, BEng
3
A Bravin, PhD
3
A L Hanson, PhD
1
M M Nawrocky, BA
1
P L Micca, BS
1
D N Slatkin, MD
1,4 and
J A Laissue, MD
4
1 Medical Department, Brookhaven National Laboratory, Upton, NY 11973-5000, USA, 2 Niederwiesstrasse 13C, Untersiggenthal, Switzerland, 3 European Synchrotron Radiation Facility, 6 Rue Jules Horowitz, BP 220, Grenoble, France and 4 Pathologisches Institut der Universität Bern, Murtenstrasse 31, Bern, Switzerland
 |
Abstract
|
|---|
Microbeam radiosurgery (MBRS), also referred to as microbeam radiation therapy (MRT), was tested at the European Synchrotron Radiation Facility (ESRF). The left tibiofibular thigh of a mouse bearing a subcutaneously (sc) implanted mouse model (SCCVII) of aggressive human squamous-cell carcinoma was irradiated in two orthogonal exposures with or without a 16 mm aluminium filter through a multislit collimator (MSC) by arrays of nearly parallel microbeams spaced 200 µm on centre (oc). The peak skin-entrance dose from each exposure was 442 Gy, 625 Gy, or 884 Gy from 35 µm wide beams or 442 Gy from 70 µm wide beams. The 442/35, 625/35, 884/35 and 442/70 MBRSs yielded 25 day, 29 day, 37 day and 35 day median survival times (MST) (post-irradiation), respectively, exceeding the 20 day MST from 35 Gy-irradiation of SCCVIIs with a seamless 100 kVp X-ray beam.
|
Introduction
|
|---|
A century ago, radiotoxic doses of X-rays delivered through a flexible grid of 1 mm thick strands of iron woven 3.5 mm on centre and a thin, continuous underlay of leather (a low-Z filter), pressed hard against the skin to blanch it, were able to palliate deep malignancies safely; iron-shielded epidermal cells healed the resultant punctate skin burns within 2 weeks [1]. After half a century, such millimetre-scale grid therapy (GT) was generally superseded by skin-sparing megavoltage radiotherapy, although at least one centre is currently pursuing a version of GT clinically [2].
It was, however, the radiobiological studies in mice, which used a deuteron microbeam to simulate cosmic radiation in space [3] that led to microbeam radiosurgery (MBRS) investigations, GT's micrometre-scale analogue. The MBRS studies have continued since 
1990 using 200–800 Gy doses of 30–200 keV X-rays delivered almost instantaneously through an array of multiple nearly parallel microslices of tissues [4–13]. Putatively, MBRS irreparably damages microsegments of neoplastic but not of normal endothelium; surviving clonogenic tumour cells may be insufficiently perfused and too sparse to re-grow.
Imminently lethal intracerebral rat 9L gliosarcomas have been palliated with 25 µm wide microbeams, 100 µm on centre (oc). About 4 months later when untreated controls had long been euthanized for tumour overgrowth, 50%, 18%, or 36% of rats remained alive after crossfired 625 Gy, crossfired 312 Gy, or unidirectional 625 Gy skin-entrance doses, respectively [7].
Despite its weak immunogenicity [14] and robust radioresistance [15], the deadly aggressive squamous-cell carcinoma (SCCVII) can be ablated either by immunotherapy [16] or by X-irradiation using a radiosensitizer [17]. However, the outcome of an experimental therapy for the murine SCCVII carcinoma is generally informative in terms of growth delay rather than ablation [18, 19]. Accordingly, we compared SCCVII growth delays and their normal-tissue radiotoxicities following different MBRS strategies to enable future ranking of various proposed clinical MBRS treatment plans.
|
Material and methods
|
|---|
Radiation source
MBRS was performed at the ID17 beamline of the European Synchrotron Radiation Facility (ESRF), a 6 GeV electron storage ring with an operating current of 180–200 mA. Beamline ID17 is equipped with a 1.6 T wiggler, which produces a beam of X-rays [20, 21] with a median energy of 38.1 keV. The beam is filtered with 1.5 m each of C and Al followed by 1.0 mm Cu. This filtration hardened the spectrum to 93 keV at maximum intensity, suitable for MBRS. The beam emerged from the beam pipe through a beryllium window to air in the radiation-shielded ID17 irradiation hutch, where it was collimated to 18 mm x 0.5 mm.
Collimator and irradiations
The microbeams were created with a variable width tungsten multislit collimator (MSC) (Tecomet, Woburn, MA) before impinging on the animal [13]. For MBRS, the anaesthetized mouse was placed prone, lengthwise, on the 15 cm x 1.5 cm surface of a 15 cm x 6.5 cm x 1.5 cm Plexiglas® block, each foreleg and the left, tumour-bearing hind leg gently taped to the sides of the block (Figure 1
). The first exposure (of the entire tumour-bearing left tibiofibular thigh) was nearly anteroposterior, with the mouse saddle rotated 5° clockwise (from the horizontal 0° reference direction of the oncoming beam) about a vertical axis (as seen by an observer looking downward toward the mouse, Figure 2) to avoid irradiating the left foreleg; the second (orthogonal) exposure was implemented after the block was rotated 95° clockwise from the 0° reference direction about the same vertical axis. Although each of the two 16 mm broad, 15 mm high anatomical (skin-entrance) targets had its estimated vertical and horizontal midplane at the estimated level of the centre of the tumour, the actual upper horizontal limit of each target was parallel to and 1 mm below the long edge of the block's upper surface. The right hind leg had been taped slightly backward to avoid exposure to microbeams during the second exposure. For each irradiation, a computer-guided platform moved the mouse directly upward (at several cm s–1) past the microbeam array emerging from the MSC. The shutter-activated exposure time was selected to conform to the slowly decaying ring current and the pre-programmed upward acceleration and speed of the platform.
View larger version (130K):
[in this window]
[in a new window]
|
Figure 1. Photograph of anaesthetized female C3H mouse bearing a leg squamous-cell carcinoma (SCCVII) carcinoma taped to Plexiglas® block, readied for microbeam radiosurgery (MBRS) at the European Synchrotron Radiation Facility (ESRF).
|
|
View larger version (6K):
[in this window]
[in a new window]
|
Figure 2. A Plexiglas® polymethylmethacrylate block (thick black outline) served as a "saddle" for the mouse, viewed as it would be by an observer directly above it. The mouse was anaesthetized and placed prone on the block for its first tumour irradiation. In this figure, the outline of the mouse is represented by an ellipse. Two black dots represent its eyes. To avoid irradiating its left foreleg, the 150 mm long axis of the block was rotated 5° (about a vertical axis through the centre of the block) clockwise from the reference 0° microbeam direction. The microbeam array, symbolized by thin arrows, was propagated in a thin, wide, slightly divergent fan-beam, substantially in a horizontal plane, represented here as the plane of this page. The second irradiation was implemented after the block was rotated 95° clockwise from the 0° reference direction about the same vertical axis.
|
|
Animal tumour model
SCCVII murine squamous cell carcinoma cells (Prof. J Martin Brown, Stanford University) were cultured in D-MEM enriched with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% L-glutamine. Only passages 1–3 were used to initiate tumours. Cells (2 x 105 in 0.05 ml of medium) were then implanted subcutaneously (sc) into the left thighs of 20–25 g female C3H mice (Taconic Farms, Germantown, NY or Charles River Laboratories, Wilmington, MA). Alternatively, freshly removed 1 mm3 fragments of mouse tumours that had been initiated sc on the dorsal thorax with 5 x 105 cells in 0.1 ml of medium [22] were minced in saline, then implanted sc in the left thighs through a 16-gauge trocar. All MBRS-irradiated tumours and 16 of the 40 untreated control tumours grew from cell suspensions. Our preliminary studies had shown that growth rates using cell suspensions were the same as those using tumour fragments; the former are preferred because the suspended cells do not seem to form satellite tumours along the trocar track when implanted. Mice bearing 80–100 mg tumours (as estimated from volume 
x2y/2, where x < y) were anaesthetized (0.01 ml per gram of body weight (gbw) of an aqueous 6 mg ml–1 sodium pentobarbital solution) by intraperitoneal injection, (60 µg gbw–1) for MBRS. Mice were irradiated 10 days after tumours were implanted. Median survival times (MST) are defined as the time interval between the day the treated groups were irradiated, which is equivalent to 10 days after tumour implantation, and the day they were euthanized unless otherwise stated.
Therapy studies
Single-exposure irradiations were used throughout. Tumour dimensions were measured 2–3 times per week and mice were euthanized either when estimated tumour volumes exceeded 500 mm3 or when skin ulceration or severe oedema (foot diameter > 5 mm) was observed. Mice were weighed whenever the tumours were measured, except during the first week after irradiation, when they were weighed daily.
100 kVp seamless X-rays
In three groups of anaesthetized mice placed prone on a horizontal surface, tumours were X-irradiated at 2.10 Gy min–1 vertically downward, delivering 25 Gy or 35 Gy. A Philips RT-100 generator was operated at 100 kVp and 8 mA with a 0.4 mm thick Cu filter, a 10 cm focus-to-skin distance, and a 2.5 cm collimator aperture in contact with the thigh. Radiation dosimetry was carried out using an air-equivalent thimble ionization chamber, adhering to the 1996 IPEB code of practice for 10–300 kVp, Cu-filtered X-rays [23].
|
Irradiation groups
|
|---|
The rapidly growing SCCVII cancers were treated in a clinically analogous way, i.e. after the tumours became palpable, which took 7 days after implantation (volumes 
ge;50 mm3). They were then sorted into groups bearing tumours of comparable size and were irradiated 3 days later, 1 day after they arrived at the ESRF.
Microbeam widths were either 35 µm or 70 µm and the on centre (oc) distances for each of the treatment groups were 200 µm. Groups 1, 2 and 3 were irradiated at skin-entrance doses of 442 Gy, 625 Gy, and 884 Gy, respectively, using 35 µm microbeam widths in each direction. Group 4 was similarly irradiated to Group 3 (884 Gy) but with a 16 mm aluminium filter upstream from the collimator. Group 5 was irradiated at a skin-entrance dose of 442 Gy with 70 µm microbeam widths in each direction and Group 6 was similarly irradiated but with the 16 mm aluminium filter. The control group comprised 40 untreated SCCVII tumour-bearing mice from five separate experiments.
|
Results
|
|---|
100 kVp seamless X-irradiation at 25 Gy and 35 Gy yielded MSTs of 14 days and 20 days, but long-term survivals were only 0/10 and 1/9, respectively (Figure 3a). Untreated controls had a MST of only 6 days or a median post-implantation survival time of 16 days.
View larger version (13K):
[in this window]
[in a new window]
|
Figure 3. Kaplan-Meier graphs of C3H mice bearing aggressive squamous-cell carcinoma (SCCVII) leg carcinomas irradiated with various radiation modalities. The on-centre distances for microbeam radiosurgery (MBRS)-irradiations were 200 µm. Mice euthanized due to foot/leg damage were not distinguished from those euthanized due to tumour overgrowth: (a) Survival graphs of mice bearing SCCVII carcinomas treated with seamless 25 Gy or 35 Gy skin-entrance doses of X-rays in comparison with unirradiated controls. (b) Survival graphs of similar mice in MBRS groups (1–6) with skin entrance doses of 442 Gy, 625 Gy, and 884 Gy at 35 µm and 442 at 70 µm beam width. "Al" designates a 16 mm-thick aluminium filter placed upstream from the collimator.
|
|
MBRS survival data are shown in Figure 3b
. Figure 4 shows average growth rates of various irradiated and control SCCVIIs. In Groups 1 and 2, euthanasia was usually for tumour overgrowth (volume ge;500 mm3); in Groups 3–6, it was mainly for foot/leg damage (severe oedema; diameter of the foot >5 mm) (Table 1). Figures 3 and 4 do not distinguish those reasons for euthanasia. Euthanasia necessitated by skin radiotoxicity probably prevented much longer survivals of the third of 884/35 and 442/70 MBRS mice that showed no residual tumour at necropsy.
View larger version (25K):
[in this window]
[in a new window]
|
Figure 4. Average relative tumour volumes of the various microbeam radiosurgery (MBRS)-irradiated and control mice. The lower tumour volumes noted in groups 3 to 6 relate to the fact that those tumours had regressed to relatively small or undetectable volumes when most of the mice had to be euthanized due to severe radiodermatitis of the inner thigh.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1. Number of ablated tumours, median survival times and explanations for euthanasia in mice treated with microbeam radiosurgery (MBRS) tracked up to 153 days after irradiation compared with those treated with seamless X-rays and with untreated controls
|
|
MBRS yielded long-term survival rates (up to 153 days) of 0/12 in Groups 1 and 2, 1/10 in Group 3, and 0/10 in Groups 4, 5, and 6. Group 3 (884/35 without aluminium) showed the highest median survival time, and only 1/10 was euthanized for tumour overgrowth; but 8/10 were euthanized for foot/leg damage and only 1 of those 8 showed no tumour at necropsy (Table 1). In contrast, in Group 5 (442/70 without aluminium), 9/10 mice were euthanized due to severe foot/leg damage, of which 4/9 euthanized mice (two each on days 27 and 31) showed no residual tumour at necropsy. On those same days (27 and 31) in Group 3, seven and four mice, respectively, had no tumours, indicating that the lower incidence of tumours in Group 5 compared with Group 3 was due to the earlier time of euthanasia.
Some non-parametric Wilcoxon Two-Sample analyses to rank palliation rates, using a morbidity/mortality index technique [24] on days 9 and 23, before foot/leg damage became apparent, are shown in Table 2. Without regard to radiodermatitis, Group 3 (884 Gy) followed by Group 5 (442/70, showed the most effective tumour palliation, which was expected from the survival graphs (Figure 3b) and because they received the highest tumour ionization energies.
View this table:
[in this window]
[in a new window]
|
Table 2. p-values from the non-parametric Wilcoxon Two-Sample Test on tumour volumes using morbidity/mortality indices [24] on days 9 and 23 after irradiationa
|
|
|
Discussion
|
|---|
Figures 3 and 4, and Table 1, demonstrate that MBRS delayed tumour growth more than did seamless 100 kVp X-rays and that the former effect is dose-dependent. However, in the higher dose groups, the plotted growth rates after day 40 are based on only a few animals, as many mice had to be euthanized due to radiodermatitis. The MSTs of each MBRS-treated group were longer than were the 14 day and 20 day MSTs observed for the seamless 25 Gy or 35 Gy groups, respectively.
Normal tissue damage occurred more quickly in mice irradiated with the broader microbeams imparting less energy per beam (442/70) than in the group with the narrower microbeam imparting greater energy per beam (884/35). The radiation field of the SCCVII carcinoma on the mouse leg encompassed the entire thigh, but not the foot (Figure 1). Radiodermatitis was most marked in the inner thigh and oedema was most severe in the left hind foot below the irradiation field. We attribute the latter to ablation of overirradiated lymphatics proximal to the foot. At the higher radiation doses, such damage limited survival time more than did tumour overgrowth.
The radiodermatitis of the inner thigh was explained with microdosimetry simulations using the MCNPX code [25]. The simulations were performed assuming a water phantom of the left mouse thigh, shaped as an inverted, truncated cone (16 mm high with a 13 mm diameter top and a 3 mm diameter bottom) in which a 0.4 mm diameter sphere of water, the phantom tumour, was embedded. Computations showed that doses between the microbeams ("valley doses") in the epidermis adjoining the Plexiglas® would have been 25% less without contributions from back-scattered X-rays. Even at 1.5 mm from the Plexiglas®, the dose would have been reduced 15% if the Plexiglas® was not present.
|
Conclusions
|
|---|
Palliation of the exceptionally radioresistant murine SCCVII carcinoma was better from MBRS than from seamless 35 Gy irradiation with no more risk to normal tissue in the radiation field. Normal-tissue damage in the higher-dose MBRS groups, especially to the left foot below the radiation field, could be deemed clinically irrelevant as most of that damage was anatomically remote from the cancer in structures that would have been spared high doses under clinical circumstances. Left foot oedema probably resulted from radiation-induced strictures of proximal blood vessels and lymphatics. Thus our computations suggest that MBRS of such SCCVII tumours using similar skin-entrance doses without the irradiated skin in contact with the Plexiglas® may enable a greater proportion of mice to survive long-term.
|
Acknowledgments
|
|---|
The authors thank Mr Seymour Brittman of Brittman & Son, East Northport, New York, for constructing the ventilated hardwood cases to enclose mouse cages for intercontinental air transportation. We also thank Mr Larry McMillan of Swiss International Airlines and his coworkers for facilitating our air travel with mice at the JFK Airport.
|
Footnotes
|
|---|
This manuscript has been sponsored by Brookhaven Science Associates, LLC under Contract No. DE-AC02-98CH10886 with the United States Department of Energy. The US Government retains, and the publisher, by accepting the article for publication, acknowledges, a world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for the US Government purposes. Funding was provided by the DOE Office of Biological and Environmental Research, the Institute of Pathology of the University of Bern, and the European Synchrotron Radiation Facility. 
Received for publication January 7, 2005.
Revision received June 2, 2005.
Accepted for publication June 16, 2005.
|
References
|
|---|
- Köhler A. Zur Röntgentiefentherapie mit Massendosen. Münchener medizinische Wochenschrift 1909;56:2314–6.
- Zwicker RD, Meigooni A, Mohiuddin M. Therapeutic advantage of grid irradiation for large single fractions. Int J Radiat Oncol Biol Phys 2004;58:1309–15.[Medline]
- Curtis HJ. The microbeam as a tool in radiobiology. Adv Biol Med Phys 1963;175:207–24.[Medline]
- Slatkin DN, Spanne P, Dilmanian FA, Sandborg M. Microbeam radiation therapy. Med Phys 1992;19:1395–400.[CrossRef][Medline]
- Slatkin DN, Dilmanian FA, Nawrocky MM, Spanne P, Gebbers J-O, Archer DW, et al. Design of a multislit, variable-width collimator for microplanar beam radiotherapy. Rev Sci Instr 1995;66:1346–7.[CrossRef]
- Slatkin DN, Spanne P, Dilmanian FA, Gebbers J-O, Laissue JA. Subacute neuropathological effects of microplanar beams of x-rays from a synchrotron wiggler. Proc Natl Acad Sci USA 1995;92:8783–7.[Abstract/Free Full Text]
- Laissue JA, Geiser G, Spanne PO, Dilmanian FA, Gebbers J-O, Geiser M, et al. Neuropathology of ablation of rat gliosarcomas and contiguous brain tissues using a microplanar beam of synchrotron-wiggler-generated X rays. Int J Cancer 1998;78:654–60.[CrossRef][Medline]
- Dilmanian FA, Morris GM, Zhong N, Bacarian T, Hainfeld JF, Kalef-Ezra J, et al. Murine EMT-6 carcinoma: high therapeutic efficacy of microbeam radiation therapy. Radiat Res 2003;159:632–41.[CrossRef][Medline]
- Schweizer PM, Spanne P, Di Michiel M, Jauch U, Blattmann H, Laissue JA. Tissue lesions caused by microplanar beams of synchrotron-generated X-rays in Drosophila melanogaster. Int J Radiat Biol 2000;76:567–74.[Medline]
- Laissue JA, Lyubimova N, Wagner H-P, Archer DW, Slatkin DN, Di Michiel M, et al. Microbeam radiation therapy, in medical applications of penetrating radiation. In: Bradford Barber H, Roehrig H, editors. Proceedings of SPIE 1999;3770:38–45.[CrossRef]
- Laissue JA, Blattmann H, Di Michiel M, Slatkin DN, Lyubimova N, Guzman R, et al. The weanling piglet cerebellum: a surrogate for tolerance to MRT (microbeam radiation therapy) in pediatric neuro-oncology. In: Bradford Barber H, Roehrig H, Doty FP, Schirato RC, Morton EJ, editors. Penetrating radiation systems and applications III. Proceedings of SPIE 2001;4508:65–73.[CrossRef]
- Blattmann H, Burkard W, Djonov V, Di Michiel M, Brauer E, Stepanek J, et al. Microbeam irradiation of the chorio-allantoic membrane (CAM) of chicken embryo. Strahlenther Onkol 2002;178:118.
- Bräuer-Krisch E, Bravin A, Zhang L, Siegbahn E, Stepanek J, Blattmann H, et al. Characterization of a tungsten/gas multislit collimator [TMSC] for microbeam radiation therapy at the European Synchrotron Radiation Facility. Rev Sci Instr 2005;76:0643031–7.
- Kanazawa H, Rapacchietta D, Kallman RF. Schedule-dependent therapeutic gain from the combination of fractionated irradiation and cis-diamminedichloroplatinum(II) in C3H/Km mouse model systems. Cancer Res 1988;48:3158–64.[Abstract/Free Full Text]
- Suit H, Allam A, Allalunis-Turner J, Brock W, Girinsky T, Hill S, et al. Is tumor cell radiation resistance correlated with metastatic ability? Cancer Res 1994;54:1736–41.[Abstract/Free Full Text]
- Mandpe AH, Tsung K, Norton JA. Cure of an established nonimmunogenic tumor, SCC VII, with a novel interleukin 12-based immunotherapy regimen in C3H mice. Arch Otolaryngol Head Neck Surg 2003;129:786–92.[Abstract/Free Full Text]
- Nakajima S, Fujii T, Murakami N, Aburano T, Sakata I, Nakae Y, et al. Therapeutic and imaging capacity of tumor-localizing radiosensitive Mn-porphyrin KADT-F10 for SCCVII tumors in C3H/He mice. Cancer Lett 2002;181:173–8.[Medline]
- Ning S, Yu N, Brown DM, Kanekal S, Knox SJ. Radiosensitization by intratumoral administration of cisplatin in a sustained-release drug delivery system. Radiother Oncol 1999;50:215–23.[CrossRef][Medline]
- Katori K, Baba Y, Imagawa Y, Nishimura G, Kagesato, Takagi E, et al. Reduction of in vivo tumor growth by MMI-166, a selective matrix metalloproteinase inhibitor, through inhibition of tumor angiogenesis in squamous cell carcinoma cell lines of head and neck. Cancer Lett 2002;178:151–9.[CrossRef][Medline]
- Thomlinson W, Berkvens P, Berruyer G, Bertrand B, Blattmann H, Bräuer-Krisch E, et al. Research at the European Synchrotron Radiation Facility medical beamline. Cell Mol Biol 2000;46:1053–63.[Medline]
- Bräuer-Krisch E, Bravin A, Lerch M, Rosenfeld A, Stepanek J, Di Michiel M, et al. MOSFET dosimetry for microbeam radiation therapy at the European Synchrotron Radiation Facility. Med Phys 2003;30:583–9.[Medline]
- Fu KK, Rayner PA, Lam KN. Modification of the effects of continuous low dose rate irradiation by concurrent chemotherapy infusion. Int J Radiat Oncol Biol Phys 1984;10:1473–8.[Medline]
- Klevenhagen SC, Aukett RJ, Harrison RM, Moretti C, Nahum AE, Rosser KE. The IPEMB code of practice for the determination of absorbed dose for x-rays below 300 kV generating potentials (0.035 mm Al-4 mm Cu HVL; 10-300 kV generating potential). Phys Med Biol 1996;41:2605–25.[CrossRef][Medline]
- Coderre JA, Slatkin DN, Micca PL, Ciallella JR. Boron neutron capture therapy of a murine melanoma with p-boronphenylalanine: dose-response analysis using a morbidity index. Radiat Res 1991;128:177–85.[Medline]
- Los Alamos National Laboratory Report LA-UR-03-5916; August, 2003.
This article has been cited by other articles:
|
|
|
|
 
F. A. Dilmanian, Z. Zhong, T. Bacarian, H. Benveniste, P. Romanelli, R. Wang, J. Welwart, T. Yuasa, E. M. Rosen, and D. J. Anschel
Interlaced x-ray microplanar beams: A radiosurgery approach with clinical potential
PNAS,
June 20, 2006;
103(25):
9709 - 9714.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
