Radiat Environ Biophys (2015) 54:335–342 DOI 10.1007/s00411-015-0600-y
ORIGINAL PAPER
The influence of the channel size on the reduction of side effects in microchannel proton therapy Stefanie Girst1 • Christoph Greubel1 • Judith Reindl1 • Christian Siebenwirth1,2 Olga Zlobinskaya2 • Gu¨nther Dollinger1 • Thomas E. Schmid2
•
Received: 28 November 2014 / Accepted: 29 April 2015 / Published online: 9 May 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Abstract The potential of proton microchannel radiotherapy to reduce radiation effects in the healthy tissue but to keep tumor control the same as in conventional proton therapy is further elucidated. The microchannels spread on their way to the tumor tissue resulting in different fractions of the healthy tissue covered with doses larger than the tumor dose, while the tumor gets homogeneously irradiated. The aim of this study was to evaluate the effect of increasing channel width on potential side effects in the normal tissue. A rectangular 180 9 180 lm2 and two Gaussian-type dose distributions of r = 260 lm and r = 520 lm with an interchannel distance of 1.8 mm have been applied by 20-MeV protons to a 3D human skin model in order to simulate the widened channels and to compare the irradiation effects at different endpoints to those of a homogeneous proton irradiation. The number of protons applied was kept constant at all irradiation modes resulting in the same average dose of 2 Gy. All kinds of proton microchannel irradiation lead to higher cell viability and produce significantly less genetic damage than homogeneous proton irradiation, but the reduction is lower for the wider channel sizes. Our findings point toward the application of microchannel irradiation for clinical proton or heavy ion therapy to further reduce damage of normal tissues while maintaining tumor control via a homogeneous dose distribution inside the tumor.
& Stefanie Girst [email protected] 1
Institut fu¨r Angewandte Physik und Messtechnik (LRT2), Universita¨t der Bundeswehr Mu¨nchen, 85579 Neubiberg, Germany
2
Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universita¨t Mu¨nchen, Munich, Germany
Keywords Radiation therapy Particle therapy Microbeam Spatial fractionation Micronuclei MTT test
Introduction The main goal of radiation therapy is to fully eradicate the tumor, while sparing the surrounding healthy tissue as much as possible (Laissue et al. 2007). Normal tissue damage or even secondary cancer induction is a major concern in radiotherapy, with detrimental effects on the patient’s well-being after tumor therapy. Proton microchannel radiotherapy, a spatially fractionated radiotherapy approach using sub-millimeter or even micrometer-sized proton beams which spread out into the tumor to homogeneously irradiate the cancer cells, was recently invented (Zlobinskaya et al. 2013). The proton microchannel approach is similar to the X-ray microbeam radiation therapy (MRT) method developed at Brookhaven National Laboratory (Slatkin et al. 1995) and the European Synchrotron Radiation Facility (ESRF; Laissue et al. 2007; Serduc et al. 2008; Brauer-Krisch et al. 2003, 2005), where side effects in the healthy tissue are substantially reduced since only a small fraction of the irradiated area suffers from large doses, but the other fraction obtains only small doses. The main difference compared to X-ray microbeam radiation therapy is that the proton microchannels spread laterally with depth inside the body. Thus, a homogeneous dose distribution and a tumor control as in normal radiation therapy are obtained when applying the proton microchannels from a single direction, while the microbeam geometry is nearly unchanged within the tumor site when applying X-ray microbeams from one direction. The only requirement is that the channel-to-channel distance is kept smaller than the proton beam lateral spread inside the
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tumor. The channel-to-channel distances can be chosen larger than 1 mm in most cases, and the side effects may be similarly or even more reduced than in the X-ray microbeam therapy where channel-to-channel distances of much smaller than 1 mm are proposed. First experimental evidence has demonstrated that a proton microchannel irradiation reduces irradiation effects in a human skin model in comparison with conventional broad-beam irradiation (Zlobinskaya et al. 2013). In this study, 20-MeV protons were applied on the central 4 9 4 mm2 of the skin in a focused microchannel mode (50-lm-wide channels on a 500 9 500 lm2 matrix) or a homogeneous mode with the same mean dose of 2 Gy at the ion microprobe SNAKE in Munich (Superconducting Nanoprobe for Applied nuclear [Kern] physics Experiments; Hauptner et al. 2004; Greubel et al. 2008; Schmid et al. 2010). MTT tissue viability test and micronucleus assay revealed higher cellular viability and lower genetic damage after microchannel irradiation compared to homogeneous broad-beam irradiation. Inflammatory response, measured via the release of inflammatory cytokines in the culture medium, was also significantly lower using the microchannel irradiation mode (Zlobinskaya et al. 2013). The overall aim of this study was to investigate the effects of proton microchannel radiotherapy on normal tissue between the skin and the tumor, where the initially micrometer-sized channels start widening with increasing depth, producing different inhomogeneous dose patterns compared to the beam entrance (see Fig. 1). This study is the continuation of our pilot study (Zlobinskaya et al. 2013) on proton microchannel radiotherapy, to fully investigate the potential of this radiotherapy method to minimize the risk of normal tissue damage in radiotherapy. For comparability of the effects, the previously used human skin model was again taken as a representative of normal tissue, to simulate the acute side effects and induced radiation damage in the deeper lying tissues. It should be noted that the radiobiological response of this tissue model
a
b
c
might not fully be representative of the response of the relevant tissues at depth, because skin is an early-reacting tissue. However, for modeling the dependency of the channel size with the radiobiological response, it seems appropriate to focus on only one representative of normal tissues to get quantitative data, which can be compared. Our research goal was the comparative analysis of tissue viability and genetic damage after proton irradiation with different sizes of increasingly widened microchannels, which represents the pathway of the protons toward the tumor. In addition, we enlarged the point-to-point distances of the irradiated matrix in order to approach dimensions that could be realistic for a microchannel proton tumor therapy in humans in future.
Materials and methods Tissue construct As in the previous study (Zlobinskaya et al. 2013), the reconstructed human skin model (EFT400; EpiDermFTTM, surface area 1 cm2) was obtained from MatTek Corporation, Ashland, MA, USA. This three-dimensional, multilayered, differentiated tissue model with an epidermal and a dermal layer consists of human-derived epidermal keratinocytes and dermal fibroblasts, cultured on special cell culture inserts. Upon arrival, the tissues were transferred to 12-well plates, each containing 2.0 ml of fresh 37 °C New Maintenance Medium (NMM, MatTek Corporation, Ashland, MA, USA), which were then incubated at 37 °C in a humidified atmosphere of 5 % CO2, replacing the culture medium every 24 h. Irradiation conditions The proton irradiations took place at the Munich ion microprobe SNAKE at the 14 MV Munich tandem accelerator, where a counted number of protons can be applied
d D= 2Gy
2 mm
Fig. 1 Dose distributions for microchannel and homogeneous irradiations. a Microchannel irradiation (SC, 180 9 180 lm2), b widened channels (WC, r * 260 lm), c overlapping channels
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0
(OC, r * 520 lm) and d homogeneous irradiation with the same average dose D = 2 Gy (doses CD are red on the color bar, e.g., maximum dose in (a) is up to 100 D)
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with micrometer precision, ensuring a precise dose application (Hauptner et al. 2004; Greubel et al. 2008; Schmid et al. 2010; Zlobinskaya et al. 2013). For the generation of the widened channels, a 200-lmthick aluminum sheet was mounted directly behind the beam exit nozzle (Fig. 2) resulting in an angular spread of the beam with nearly Gaussian angle distribution. In an air gap between the aluminum sheet and the sample, this angular spread is transformed into a lateral spread of Gaussian shape, which width can be adjusted by the length of the air gap, i.e., the distance between sample and Al. For comparability with our previous study (Zlobinskaya et al. 2013) where 20-MeV protons had been used, protons were accelerated to 21 MeV to account for the energy loss in the 200-lm aluminum resulting in a 20-MeV proton beam behind [cf. SRIM (Ziegler and Biersack 2003)]. Four different irradiation modes were produced, all using the same total number of protons (3.82 9 108 p) and thus mean dose of 2 Gy (±5 %, cf. Zlobinskaya et al. 2013), but with different channel sizes and dose distributions. Three different channel irradiations and one homogeneous dose distribution (‘‘homogeneous field’’ HF, Fig. 1d) were applied. All channel irradiations consisted of a matrix irradiation that was enlarged to a 1.8 9 1.8 mm2 matrix compared to the original study (Zlobinskaya et al. 2013) in order to consider an irradiation mode close to a hypothetic therapy arrangement. The matrix points differed in the irradiated spot size for the three-channel irradiation modes adjusting the spot size by the distance of the sample from the aluminum plate. The smallest spots were achieved by scanning a 1-lm-sized beam to a square size of 180 9 180 lm2 and placing the tissue sample directly behind the aluminum sheet. Thus, these microchannels (‘‘small channels’’ SC, Fig. 1a) covered about 1 % of the total area of the square matrix of 1.8 9 1.8 mm2 resulting in a dose of *200 Gy in the channels. The two widened modes were chosen as one would obtain in one and two-
variable distance
2
beam
Al beam exit nozzle
sample
Fig. 2 Irradiation setup at SNAKE. Mounting of a 200-lm aluminum sheet directly behind the beam exit nozzle to produce channels with Gaussian distribution of variable size r, by increasing the distance between sample and Al
thirds of the tumor depth of a model tumor at 10–15 cm depth, to evaluate the effect of the increasing channel width toward the tumor. The enlarged channels were obtained by placing the tissue samples at distances of 19 mm [‘‘widened channels’’ WC (Fig. 1b)] and 38 mm [‘‘overlapping channels’’ OC (Fig. 1c)] behind the Al plate without scanning the 1-lm spot. This resulted in nearly Gaussian-shaped dose distributions that had a standard deviation of r * 260 lm for the WC mode, as in 1/3 of the tumor depth, while the OC mode, as in 2/3 of the tumor depth, were about twice as wide, r * 520 lm. This corresponds to about 25 and 38 % of the total area irradiated with a dose equal or higher than the homogeneous dose of 2 Gy, respectively (cf. Table 1). For the homogeneous irradiation, the microbeam was scanned to 0.5 9 0.5 mm2 in front of the Al plate and put together to a homogeneous field with the same total area as the channel matrices. For the micronucleus endpoint, this was a circular area of 6 mm diameter, for the MTT endpoint, a square field of edge length 5.4 mm. During irradiation, the skin sample was fixed in a specially designed container between two 6-lm Mylar foils (Zlobinskaya et al. 2013; Schmid et al. 2010) and mounted in the defined distance behind the aluminum sheet, with the dermis facing the beam. For an exact dose calculation, every proton was detected in a scintillator–photomultiplier detector after traversing the skin sample (Zlobinskaya et al. 2013). Three skin samples were used for each irradiation mode, and the whole experiment was performed in duplicate in two independent beam times. MTT tissue viability assay The MTT assay (MTT-100, MatTek Corporation) is a colorimetric assay for the quantification of the metabolic activity of enzymes in the nucleus of the cell. The MTT substrate is only cleaved by active mitochondria, and the amount of formazan generated in this enzymatic reaction correlates closely with the cell number. In brief, 10 days after irradiation, the tissue samples were washed twice with PBS (phosphate-buffered saline; Sigma-Aldrich, Germany) and the central part of the irradiated area was cut using a 4-mm biopsy punch and placed in 300 ll/well of MTT solution. After 3 h of incubation at 37 °C, the tissues were transferred into fresh 6-well plates with 2 ml/well of extraction solution. After overnight extraction at room temperature, the tissues were removed and 200 ll of the remaining, thoroughly mixed sample was transferred into 96-well plates. The optical density (OD) of the samples was read at 570 nm using a photospectrometer, subtracting background readings at 650 nm. The percentage viability was determined for each tissue by comparison with untreated controls (cf. Zlobinskaya et al. 2013).
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338 Table 1 Fraction of total area irradiated with doses C2 Gy for the four irradiation modes
Radiat Environ Biophys (2015) 54:335–342
Fraction of total area irradiated with D C 2 Gy (%)
SC
WC
OC
HF
11 ± 2
25 ± 3
38 ± 4
100
Micronucleus assay of skin keratinocytes The micronucleus test was performed as described by Schmid (Schmid et al. 2010; Zlobinskaya et al. 2013). In brief, 3 lg/ml cytochalasin B (Sigma-Aldrich, Germany) was added into the culture medium immediately after irradiation, and the tissues were incubated for 48 h at 37 °C in a humidified atmosphere of 5 % CO2 in air. After manual separation of the tissue samples from the supporting membrane, the dermis was separated from the keratinocyte-containing epidermis. For trypsinization, each tissue was placed in 1 ml trypsin/EDTA for 20 min at 37 °C. After 3-min hypotonic treatment using 75 mM KCL, the keratinocytes were fixed in methanol/acetic acid (3:1) and microscope slides were prepared and stained with acridine orange (20 lg/ml). The frequency of MN could be counted precisely in the first division cycle after irradiation, identified from the binucleate appearance of the divided cells due to the cytokinesis block (CB). For each sample, 500 binucleated CB cells with well-preserved cytoplasm containing detached MN were analyzed. The dose modification factor, DMF, is defined, in the same way as the relative biological effectiveness, as the ratio of the dose of a reference radiation, Dref, and the test radiation, Dtest, to induce the same effect. For the determination of dose modification factors (DMFs), dose–response curves of reference radiation (200-kV X-rays) were used (Zlobinskaya et al. 2013). The necessary dose of reference radiation is determined by inverting the fitted dose effect curve, y(D), with linear dependence, y(D) = c ? aD, with c = (0.0107 ± 0.0021) Gy-1 and a = (0.0247 ± 0.0020) (Zlobinskaya et al. 2013). As the parameters, a and c, are correlated with each other (correlation coefficient cor(a,c) = -0.535) for calculating the standard errors of the DMF, not only the parameter errors were taken into account but also the correlation.
Results Dose distributions for microchannels and widened channels Radiochromic Gafchromic films (Gafchromic EBT2) were used for visualization and quantitative analysis of the dose distributions obtained from the various channel irradiations (Reinhardt et al. 2012). The irradiation pattern on the Gafchromic films clearly demonstrated the channel
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Fig. 3 Dose distribution of the microchannel irradiation mode (SC). X and y projections of the dose values of the quadratic field are displayed as fractions of the mean dose (i.e., the dose averaged over the total area) on a logarithmic scale. The data show a dose falloff below 1 % of the mean dose at a distance of approximately 210 lm from the border of the irradiated field (film polymers *20 lm, scanner resolution *21 lm). This means that *89 % of the area contains less than 1 % of the average dose, i.e., less than 0.02 Gy. The arrow indicates the saturation of the Gafchromic film within the irradiated 180 9 180 lm2 field where the dose values are 100 times the mean dose (2000 Gy)
character of the irradiation, revealing a sharp dose drop at the borders of the irradiated microchannels. In order to quantify the valley dose between the microchannels, ten times the number of protons normally applied (i.e., 10 9 1.53 9 107 = 1.53 9 108) were delivered to one 180 9 180 lm2 field to obtain valley doses in the sensitivity range of the Gafchromic films. The data shown in Fig. 3 reveal that at least 89 % of the area receives less than 1 % of the applied mean dose (i.e., less than 0.02 Gy) in the microchannel mode. The slightly higher valley dose in comparison with (Zlobinskaya et al. 2013) arises from large-angle scattering events of protons within the aluminum sheet. For the characterization of the widened channel modes, calibration curves for different distances between the aluminum sheet and the Gafchromic film were recorded for the three independent experiments. The width, r, of the resulting Gaussian-shaped proton distributions is plotted versus the corresponding air gap in Fig. 4, showing a linear dependence. The standard deviation r of the WC and OC mode for the three experiments is given in Table 2, with a mean and standard deviation of (258 ± 11) lm in the WC
Radiat Environ Biophys (2015) 54:335–342
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mode and a channel width of (514 ± 10) lm in the OC mode. MTT tissue cytotoxicity test Cellular viability was measured 10 days after irradiation via the colorimetric MTT cytotoxicity assay. Pooled mean values and the standard errors of two independent experiments (Exp. 2 ? 3) are shown in Fig. 5, and data from each experiment are given in Table 3. Homogeneous irradiation (HF) reduced cell viability to 33 ± 3 % compared to unirradiated controls, while microchannel irradiation resulted in 85 ± 10 % cell viability. The widened channels lead to tissue viabilities of 75 ± 6 % (WC) and 74 ± 7 % (OC), respectively. The resulting values for homogeneous irradiation are significantly lower than all the three channel modes (P \ 0.05). The values of SC, WC and OC, however, were not significantly different using the unpaired t test.
WC, OC and HF), with three skin samples each in two separate experiments (Exp. 1 ? 2). The pooled data of the two independent experiments are given as mean values in the tables and shown in Fig. 6. The X-ray reference curve, obtained from irradiation with 200-kV X-rays, was already published in (Zlobinskaya et al. 2013). In order to quantify the genetic damage of the four irradiation modes compared to a homogeneous X-ray irradiation, a dose modification factor (DMF), similar to the RBE, was calculated, using the dose of the reference radiation 200-kV X-rays that produced a response equal to 2 Gy of protons. For the (nearly) homogeneous irradiation mode, the resulting pooled DMFHF was 1.21 ± 0.21. For microchannel irradiation, the resulting pooled DMFSC was 0.29 ± 0.11, and for the widened channels, DMFWC
Micronucleus test Tables 4 and 5 present the detailed results of micronucleus induction in binucleate keratinocytes after proton irradiation, determined at four different irradiation modes (SC,
Fig. 5 MTT test results. Cell viability of microchannel (SC), widened channel (WC), overlapping channel (OC) and homogeneous field (HF) irradiations was measured 10 day after irradiation relative to sham-treated, unirradiated controls (CO) by the MTT assay. Error bars represent the SD of the mean value of six independently irradiated EpiDermFTTM tissue samples, from two different experiments
Table 3 MTT test results of experiment 2 and 3
Fig. 4 Measured width of the resulting proton distributions at different distances behind the aluminum sheet. For the three independent experiments, Gaussian distributions at 4.5, 9, 19 and 38 mm behind the Al were analyzed with Gafchromic films
Viability (%)
Experiment 2
Experiment 3
Pooled
SC
72.9 ± 2.0
98.1 ± 1.9
85.5 ± 10.0
WC
67.8 ± 1.4
75.6 ± 1.5
74.6 ± 5.1
OC
64.3 ± 2.2
81.5 ± 1.9
74.2 ± 6.8
HF
33.4 ± 1.0
32.2 ± 1.9
32.8 ± 2.9
Table 2 Measured width r for the widened channels in the three independent experiments r (lm)
Experiment 1
Experiment 2
Experiment 3
Mean ± SD
WC
267 ± 7
261 ± 7
247 ± 7
258 ± 11
OC
520 ± 15
520 ± 15
503 ± 15
514 ± 10
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Radiat Environ Biophys (2015) 54:335–342
Table 4 Frequency and distribution of micronuclei (MN) in cytokinesis-blocked (CB) binucleated epidermal keratinocytes from skin tissue induced by 2 Gy of 20 MeV protons at four spatial irradiation modes ID number
Group
Analyzed cells
Micronuclei per CB cell y (±SE)
Intercellular distribution of micronuclei
Dispersion ratio
0
r2/y
1
2
3
1
HF
465
0.075 ± 0.017
441
15
7
2
1.67
2
HF
483
0.093 ± 0.019
455
14
11
3
1.80
3 P G1–G3
HF HF
505 1453
0.050 ± 0.013 0.072 ± 0.009
489 1385
9 38
5 23
2 7
1.79 1.77
4
SC
440
0.025 ± 0.011
434
2
3
1
2.07
5
SC
493
0.034 ± 0.012
483
5
3
2
2.03
6 P G4–G6
SC
503
0.026 ± 0.008
492
9
2
0
1.28
SC
1436
0.029 ± 0.006
1409
16
8
3
1.80
7
WC
500
0.046 ± 0.012
484
10
5
1
1.65
8
WC
496
0.040 ± 0.012
483
8
3
2
1.86
9 P 7–9
WC
466
0.054 ± 0.015
450
10
3
3
1.91
WC
1462
0.047 ± 0.008
1417
28
11
6
1.81
10
OC
510
0.049 ± 0.013
493
10
6
1
1.67
11
OC
459
0.046 ± 0.015
448
5
2
4
2.29
12 P 10–12
OC
429
0.049 ± 0.013
413
12
3
1
1.53
OC
1398
0.048 ± 0.008
1354
27
11
10
1.82
Results from experiment 1 Table 5 Frequency and distribution of micronuclei (MN) in cytokinesis-blocked (CB) binucleated epidermal keratinocytes from skin tissue induced by 2 Gy of 20 MeV protons at four spatial irradiation modes ID number
Group
Analyzed cells
Micronuclei per CB cell y (±SE)
Intercellular distribution of micronuclei
Dispersion ratio
0
1
2
3
r2/y
1
HF
508
0.075 ± 0.016
484
11
12
1
1.718
2
HF
518
0.070 ± 0.015
492
17
8
1
1.545
3 P G1–G3
HF HF
495 1521
0.061 ± 0.016 0.068 ± 0.009
477 1453
10 38
4 24
4 6
2.010 1.740
4
SC
510
0.026 ± 0.008
498
11
1
0
1.130
5
SC
460
0.013 ± 0.007
456
2
2
0
1.663
6 P G4–G6
SC
502
0.028 ± 0.011
493
6
1
2
1.975
SC
1472
0.022 ± 0.005
1447
19
4
2
1.586
7
WC
509
0.024 ± 0.009
500
7
1
1
1.645
8
WC
298
0.057 ± 0.017
285
9
4
0
1.417
9 P 7–9
WC
502
0.038 ± 0.011
486
14
1
1
1.386
WC
1309
0.037 ± 0.007
1271
30
6
2
1.463
10
OC
502
0.054 ± 0.014
484
11
5
2
1.764
11
OC
517
0.070 ± 0.015
491
17
8
1
1.545
12 P 10–12
OC
516
0.043 ± 0.013
502
8
4
2
1.872
OC
1535
0.055 ± 0.008
1477
36
17
5
1.698
Results from experiment 2
was 0.64 ± 0.14 and DMFOC was 0.93 ± 0.17. The micronucleus frequency was significantly reduced compared to the homogeneous irradiation for the microchannel mode (SC) and the widened channels (WC) (P \ 0.01), but not for the overlapping channels (OC) (P [ 0.05).
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Discussion The aim of this study was to follow up on our pilot study on proton microchannel radiotherapy (Zlobinskaya et al. 2013) with regard to the so-called intermediate region
Radiat Environ Biophys (2015) 54:335–342
Fig. 6 Micronucleus (MN) induction in cytokinesis-blocked (CB) binucleate keratinocytes from EpiDermFTTM tissues. Linear dose– response curve for 200-kV X-rays (open circles, cf. Zlobinskaya et al. 2013) and mean MN frequency for homogeneous (HF) and microchannel irradiation modes (SC, WC, OC). Uncertainty bars represent the SD of the mean value of six tissue samples, from two different experiments
between the skin and the tumor, where the microchannels widen with increasing depth in tissue, resulting in an inhomogeneous dose distribution. This is an important step for a complete evaluation of the potential of proton microchannel irradiations for radiotherapy, after the benefits of this method for normal tissue have been demonstrated earlier (Zlobinskaya et al. 2013). The increasing channel size with depth is inherent for microchannel particle therapy due to small-angle scattering effects and is used to obtain a homogeneous dose distribution inside the tumor. It is important to note that widening of the channels is smaller for heavier ions than protons, but might be enhanced for all kinds of ions by an additional beam divergence. This widening has never been investigated in the context of X-ray microbeam radiation therapy (MRT), where arrays of microscopically thin and nearly parallel synchrotrongenerated X-ray beams are used (Serduc et al. 2008; Laissue et al. 2007; Brauer-Krisch et al. 2003, 2005). Typical sizes of the microplanar beams in MRT are 25–75 lm, separated by distances of 50–400 lm. This radiotherapy method, which has been examined in several animal studies (Anschel et al. 2010), maintains the microarray/channel geometry within the tumor as X-ray beams barely spread in depth and has been shown to prevent normal tissue necrosis, especially of the brain through sparing of normal brain vessels, while efficiently ensuring tumor control. In this study, the acute radiation effects and the induced genetic damage have been investigated for beam sizes that would be obtained in the deeper lying tissues when applying microchannel proton therapy. The irradiation effects
341
in the well-established human skin model (EpiDermFTTM, EFT400, MatTek) are compared for the different beam sizes and a homogeneous proton irradiation, and the experiments may serve as a representative for normal tissue effects. The comparative analysis shows no disadvantages of microchannel radiotherapy compared to conventional homogeneous broad-beam irradiations in the intermediate region, but argues for microchannel irradiation with regard to tissue viability and genetic damage even there. Cytotoxicity could be decreased by about a factor of two for all sizes of microchannels compared to homogeneous irradiation. Even though an increasing number of micronuclei per divided cell, corresponding to higher radiation damage, is induced with increasing channel size, there is still a significant reduction of genetic damage for the widened channels (as in 1/3 of the tumor depth) compared to the homogeneous irradiation. Also for the almost homogeneously overlapping channels (as in 2/3 of the tumor depth), the genetic damage does not exceed that of a homogeneous irradiation. These findings support and confirm the conclusion of our first study (Zlobinskaya et al. 2013) that proton microchannel radiotherapy leads to reduced acute and genotoxic effects compared to conventional broad-beam irradiation but keeping the homogeneous dose distribution inside the tumor and thus the tumor control as in conventional tumor therapy. Therefore, it could be an option in clinical proton and/or heavy ion therapy that would allow reduction of side effects keeping the same tumor control or enhancing tumor dose and thus improving tumor control in radiation resistant tumor tissues at still acceptable side effects. Acknowledgments This work was supported by the DFG Cluster of Excellence ‘‘Munich-Centre for Advanced Photonics,’’ by EU FP7 DoReMi Network of Excellence and by the Maier Leibnitz Laboratory Munich. We thank Sabine Reinhardt for her support with the Gafchromic film dosimetry and the staff of MLL operating the tandem accelerator.
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Radiat Environ Biophys (2015) 54:335–342 micronucleus induction in 3D human reconstructed skin tissue. Radiother Oncol 95(1):66–72. doi:10.1016/j.radonc.2010.03.010 Serduc R, Christen T, Laissue J, Farion R, Bouchet A, Sanden B, Segebarth C, Brauer-Krisch E, Le Duc G, Bravin A, Remy C, Barbier EL (2008) Brain tumor vessel response to synchrotron microbeam radiation therapy: a short-term in vivo study. Phys Med Biol 53(13):3609–3622. doi:10.1088/0031-9155/53/13/015 Slatkin DN, Spanne P, Dilmanian FA, Gebbers JO, Laissue JA (1995) Subacute neuropathological effects of microplanar beams of X-rays from a synchrotron wiggler. Proc Natl Acad Sci USA 92(19):8783–8787 Ziegler JF, Biersack JP (2003) The stopping and range of ions in matter (SRIM2003) [online]. http://www.srim.org Zlobinskaya O, Girst S, Greubel C, Hable V, Siebenwirth C, Walsh DWM, Multhoff G, Wilkens JJ, Schmid TE, Dollinger G (2013) Reduced side effects by proton microchannel radiotherapy: study in a human skin model. Radiat Environ Biophys 52:123–133. doi:10.1007/s00411-012-0450-9
ORIGINAL PAPER
The influence of the channel size on the reduction of side effects in microchannel proton therapy Stefanie Girst1 • Christoph Greubel1 • Judith Reindl1 • Christian Siebenwirth1,2 Olga Zlobinskaya2 • Gu¨nther Dollinger1 • Thomas E. Schmid2
•
Received: 28 November 2014 / Accepted: 29 April 2015 / Published online: 9 May 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Abstract The potential of proton microchannel radiotherapy to reduce radiation effects in the healthy tissue but to keep tumor control the same as in conventional proton therapy is further elucidated. The microchannels spread on their way to the tumor tissue resulting in different fractions of the healthy tissue covered with doses larger than the tumor dose, while the tumor gets homogeneously irradiated. The aim of this study was to evaluate the effect of increasing channel width on potential side effects in the normal tissue. A rectangular 180 9 180 lm2 and two Gaussian-type dose distributions of r = 260 lm and r = 520 lm with an interchannel distance of 1.8 mm have been applied by 20-MeV protons to a 3D human skin model in order to simulate the widened channels and to compare the irradiation effects at different endpoints to those of a homogeneous proton irradiation. The number of protons applied was kept constant at all irradiation modes resulting in the same average dose of 2 Gy. All kinds of proton microchannel irradiation lead to higher cell viability and produce significantly less genetic damage than homogeneous proton irradiation, but the reduction is lower for the wider channel sizes. Our findings point toward the application of microchannel irradiation for clinical proton or heavy ion therapy to further reduce damage of normal tissues while maintaining tumor control via a homogeneous dose distribution inside the tumor.
& Stefanie Girst [email protected] 1
Institut fu¨r Angewandte Physik und Messtechnik (LRT2), Universita¨t der Bundeswehr Mu¨nchen, 85579 Neubiberg, Germany
2
Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universita¨t Mu¨nchen, Munich, Germany
Keywords Radiation therapy Particle therapy Microbeam Spatial fractionation Micronuclei MTT test
Introduction The main goal of radiation therapy is to fully eradicate the tumor, while sparing the surrounding healthy tissue as much as possible (Laissue et al. 2007). Normal tissue damage or even secondary cancer induction is a major concern in radiotherapy, with detrimental effects on the patient’s well-being after tumor therapy. Proton microchannel radiotherapy, a spatially fractionated radiotherapy approach using sub-millimeter or even micrometer-sized proton beams which spread out into the tumor to homogeneously irradiate the cancer cells, was recently invented (Zlobinskaya et al. 2013). The proton microchannel approach is similar to the X-ray microbeam radiation therapy (MRT) method developed at Brookhaven National Laboratory (Slatkin et al. 1995) and the European Synchrotron Radiation Facility (ESRF; Laissue et al. 2007; Serduc et al. 2008; Brauer-Krisch et al. 2003, 2005), where side effects in the healthy tissue are substantially reduced since only a small fraction of the irradiated area suffers from large doses, but the other fraction obtains only small doses. The main difference compared to X-ray microbeam radiation therapy is that the proton microchannels spread laterally with depth inside the body. Thus, a homogeneous dose distribution and a tumor control as in normal radiation therapy are obtained when applying the proton microchannels from a single direction, while the microbeam geometry is nearly unchanged within the tumor site when applying X-ray microbeams from one direction. The only requirement is that the channel-to-channel distance is kept smaller than the proton beam lateral spread inside the
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336
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tumor. The channel-to-channel distances can be chosen larger than 1 mm in most cases, and the side effects may be similarly or even more reduced than in the X-ray microbeam therapy where channel-to-channel distances of much smaller than 1 mm are proposed. First experimental evidence has demonstrated that a proton microchannel irradiation reduces irradiation effects in a human skin model in comparison with conventional broad-beam irradiation (Zlobinskaya et al. 2013). In this study, 20-MeV protons were applied on the central 4 9 4 mm2 of the skin in a focused microchannel mode (50-lm-wide channels on a 500 9 500 lm2 matrix) or a homogeneous mode with the same mean dose of 2 Gy at the ion microprobe SNAKE in Munich (Superconducting Nanoprobe for Applied nuclear [Kern] physics Experiments; Hauptner et al. 2004; Greubel et al. 2008; Schmid et al. 2010). MTT tissue viability test and micronucleus assay revealed higher cellular viability and lower genetic damage after microchannel irradiation compared to homogeneous broad-beam irradiation. Inflammatory response, measured via the release of inflammatory cytokines in the culture medium, was also significantly lower using the microchannel irradiation mode (Zlobinskaya et al. 2013). The overall aim of this study was to investigate the effects of proton microchannel radiotherapy on normal tissue between the skin and the tumor, where the initially micrometer-sized channels start widening with increasing depth, producing different inhomogeneous dose patterns compared to the beam entrance (see Fig. 1). This study is the continuation of our pilot study (Zlobinskaya et al. 2013) on proton microchannel radiotherapy, to fully investigate the potential of this radiotherapy method to minimize the risk of normal tissue damage in radiotherapy. For comparability of the effects, the previously used human skin model was again taken as a representative of normal tissue, to simulate the acute side effects and induced radiation damage in the deeper lying tissues. It should be noted that the radiobiological response of this tissue model
a
b
c
might not fully be representative of the response of the relevant tissues at depth, because skin is an early-reacting tissue. However, for modeling the dependency of the channel size with the radiobiological response, it seems appropriate to focus on only one representative of normal tissues to get quantitative data, which can be compared. Our research goal was the comparative analysis of tissue viability and genetic damage after proton irradiation with different sizes of increasingly widened microchannels, which represents the pathway of the protons toward the tumor. In addition, we enlarged the point-to-point distances of the irradiated matrix in order to approach dimensions that could be realistic for a microchannel proton tumor therapy in humans in future.
Materials and methods Tissue construct As in the previous study (Zlobinskaya et al. 2013), the reconstructed human skin model (EFT400; EpiDermFTTM, surface area 1 cm2) was obtained from MatTek Corporation, Ashland, MA, USA. This three-dimensional, multilayered, differentiated tissue model with an epidermal and a dermal layer consists of human-derived epidermal keratinocytes and dermal fibroblasts, cultured on special cell culture inserts. Upon arrival, the tissues were transferred to 12-well plates, each containing 2.0 ml of fresh 37 °C New Maintenance Medium (NMM, MatTek Corporation, Ashland, MA, USA), which were then incubated at 37 °C in a humidified atmosphere of 5 % CO2, replacing the culture medium every 24 h. Irradiation conditions The proton irradiations took place at the Munich ion microprobe SNAKE at the 14 MV Munich tandem accelerator, where a counted number of protons can be applied
d D= 2Gy
2 mm
Fig. 1 Dose distributions for microchannel and homogeneous irradiations. a Microchannel irradiation (SC, 180 9 180 lm2), b widened channels (WC, r * 260 lm), c overlapping channels
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0
(OC, r * 520 lm) and d homogeneous irradiation with the same average dose D = 2 Gy (doses CD are red on the color bar, e.g., maximum dose in (a) is up to 100 D)
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with micrometer precision, ensuring a precise dose application (Hauptner et al. 2004; Greubel et al. 2008; Schmid et al. 2010; Zlobinskaya et al. 2013). For the generation of the widened channels, a 200-lmthick aluminum sheet was mounted directly behind the beam exit nozzle (Fig. 2) resulting in an angular spread of the beam with nearly Gaussian angle distribution. In an air gap between the aluminum sheet and the sample, this angular spread is transformed into a lateral spread of Gaussian shape, which width can be adjusted by the length of the air gap, i.e., the distance between sample and Al. For comparability with our previous study (Zlobinskaya et al. 2013) where 20-MeV protons had been used, protons were accelerated to 21 MeV to account for the energy loss in the 200-lm aluminum resulting in a 20-MeV proton beam behind [cf. SRIM (Ziegler and Biersack 2003)]. Four different irradiation modes were produced, all using the same total number of protons (3.82 9 108 p) and thus mean dose of 2 Gy (±5 %, cf. Zlobinskaya et al. 2013), but with different channel sizes and dose distributions. Three different channel irradiations and one homogeneous dose distribution (‘‘homogeneous field’’ HF, Fig. 1d) were applied. All channel irradiations consisted of a matrix irradiation that was enlarged to a 1.8 9 1.8 mm2 matrix compared to the original study (Zlobinskaya et al. 2013) in order to consider an irradiation mode close to a hypothetic therapy arrangement. The matrix points differed in the irradiated spot size for the three-channel irradiation modes adjusting the spot size by the distance of the sample from the aluminum plate. The smallest spots were achieved by scanning a 1-lm-sized beam to a square size of 180 9 180 lm2 and placing the tissue sample directly behind the aluminum sheet. Thus, these microchannels (‘‘small channels’’ SC, Fig. 1a) covered about 1 % of the total area of the square matrix of 1.8 9 1.8 mm2 resulting in a dose of *200 Gy in the channels. The two widened modes were chosen as one would obtain in one and two-
variable distance
2
beam
Al beam exit nozzle
sample
Fig. 2 Irradiation setup at SNAKE. Mounting of a 200-lm aluminum sheet directly behind the beam exit nozzle to produce channels with Gaussian distribution of variable size r, by increasing the distance between sample and Al
thirds of the tumor depth of a model tumor at 10–15 cm depth, to evaluate the effect of the increasing channel width toward the tumor. The enlarged channels were obtained by placing the tissue samples at distances of 19 mm [‘‘widened channels’’ WC (Fig. 1b)] and 38 mm [‘‘overlapping channels’’ OC (Fig. 1c)] behind the Al plate without scanning the 1-lm spot. This resulted in nearly Gaussian-shaped dose distributions that had a standard deviation of r * 260 lm for the WC mode, as in 1/3 of the tumor depth, while the OC mode, as in 2/3 of the tumor depth, were about twice as wide, r * 520 lm. This corresponds to about 25 and 38 % of the total area irradiated with a dose equal or higher than the homogeneous dose of 2 Gy, respectively (cf. Table 1). For the homogeneous irradiation, the microbeam was scanned to 0.5 9 0.5 mm2 in front of the Al plate and put together to a homogeneous field with the same total area as the channel matrices. For the micronucleus endpoint, this was a circular area of 6 mm diameter, for the MTT endpoint, a square field of edge length 5.4 mm. During irradiation, the skin sample was fixed in a specially designed container between two 6-lm Mylar foils (Zlobinskaya et al. 2013; Schmid et al. 2010) and mounted in the defined distance behind the aluminum sheet, with the dermis facing the beam. For an exact dose calculation, every proton was detected in a scintillator–photomultiplier detector after traversing the skin sample (Zlobinskaya et al. 2013). Three skin samples were used for each irradiation mode, and the whole experiment was performed in duplicate in two independent beam times. MTT tissue viability assay The MTT assay (MTT-100, MatTek Corporation) is a colorimetric assay for the quantification of the metabolic activity of enzymes in the nucleus of the cell. The MTT substrate is only cleaved by active mitochondria, and the amount of formazan generated in this enzymatic reaction correlates closely with the cell number. In brief, 10 days after irradiation, the tissue samples were washed twice with PBS (phosphate-buffered saline; Sigma-Aldrich, Germany) and the central part of the irradiated area was cut using a 4-mm biopsy punch and placed in 300 ll/well of MTT solution. After 3 h of incubation at 37 °C, the tissues were transferred into fresh 6-well plates with 2 ml/well of extraction solution. After overnight extraction at room temperature, the tissues were removed and 200 ll of the remaining, thoroughly mixed sample was transferred into 96-well plates. The optical density (OD) of the samples was read at 570 nm using a photospectrometer, subtracting background readings at 650 nm. The percentage viability was determined for each tissue by comparison with untreated controls (cf. Zlobinskaya et al. 2013).
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338 Table 1 Fraction of total area irradiated with doses C2 Gy for the four irradiation modes
Radiat Environ Biophys (2015) 54:335–342
Fraction of total area irradiated with D C 2 Gy (%)
SC
WC
OC
HF
11 ± 2
25 ± 3
38 ± 4
100
Micronucleus assay of skin keratinocytes The micronucleus test was performed as described by Schmid (Schmid et al. 2010; Zlobinskaya et al. 2013). In brief, 3 lg/ml cytochalasin B (Sigma-Aldrich, Germany) was added into the culture medium immediately after irradiation, and the tissues were incubated for 48 h at 37 °C in a humidified atmosphere of 5 % CO2 in air. After manual separation of the tissue samples from the supporting membrane, the dermis was separated from the keratinocyte-containing epidermis. For trypsinization, each tissue was placed in 1 ml trypsin/EDTA for 20 min at 37 °C. After 3-min hypotonic treatment using 75 mM KCL, the keratinocytes were fixed in methanol/acetic acid (3:1) and microscope slides were prepared and stained with acridine orange (20 lg/ml). The frequency of MN could be counted precisely in the first division cycle after irradiation, identified from the binucleate appearance of the divided cells due to the cytokinesis block (CB). For each sample, 500 binucleated CB cells with well-preserved cytoplasm containing detached MN were analyzed. The dose modification factor, DMF, is defined, in the same way as the relative biological effectiveness, as the ratio of the dose of a reference radiation, Dref, and the test radiation, Dtest, to induce the same effect. For the determination of dose modification factors (DMFs), dose–response curves of reference radiation (200-kV X-rays) were used (Zlobinskaya et al. 2013). The necessary dose of reference radiation is determined by inverting the fitted dose effect curve, y(D), with linear dependence, y(D) = c ? aD, with c = (0.0107 ± 0.0021) Gy-1 and a = (0.0247 ± 0.0020) (Zlobinskaya et al. 2013). As the parameters, a and c, are correlated with each other (correlation coefficient cor(a,c) = -0.535) for calculating the standard errors of the DMF, not only the parameter errors were taken into account but also the correlation.
Results Dose distributions for microchannels and widened channels Radiochromic Gafchromic films (Gafchromic EBT2) were used for visualization and quantitative analysis of the dose distributions obtained from the various channel irradiations (Reinhardt et al. 2012). The irradiation pattern on the Gafchromic films clearly demonstrated the channel
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Fig. 3 Dose distribution of the microchannel irradiation mode (SC). X and y projections of the dose values of the quadratic field are displayed as fractions of the mean dose (i.e., the dose averaged over the total area) on a logarithmic scale. The data show a dose falloff below 1 % of the mean dose at a distance of approximately 210 lm from the border of the irradiated field (film polymers *20 lm, scanner resolution *21 lm). This means that *89 % of the area contains less than 1 % of the average dose, i.e., less than 0.02 Gy. The arrow indicates the saturation of the Gafchromic film within the irradiated 180 9 180 lm2 field where the dose values are 100 times the mean dose (2000 Gy)
character of the irradiation, revealing a sharp dose drop at the borders of the irradiated microchannels. In order to quantify the valley dose between the microchannels, ten times the number of protons normally applied (i.e., 10 9 1.53 9 107 = 1.53 9 108) were delivered to one 180 9 180 lm2 field to obtain valley doses in the sensitivity range of the Gafchromic films. The data shown in Fig. 3 reveal that at least 89 % of the area receives less than 1 % of the applied mean dose (i.e., less than 0.02 Gy) in the microchannel mode. The slightly higher valley dose in comparison with (Zlobinskaya et al. 2013) arises from large-angle scattering events of protons within the aluminum sheet. For the characterization of the widened channel modes, calibration curves for different distances between the aluminum sheet and the Gafchromic film were recorded for the three independent experiments. The width, r, of the resulting Gaussian-shaped proton distributions is plotted versus the corresponding air gap in Fig. 4, showing a linear dependence. The standard deviation r of the WC and OC mode for the three experiments is given in Table 2, with a mean and standard deviation of (258 ± 11) lm in the WC
Radiat Environ Biophys (2015) 54:335–342
339
mode and a channel width of (514 ± 10) lm in the OC mode. MTT tissue cytotoxicity test Cellular viability was measured 10 days after irradiation via the colorimetric MTT cytotoxicity assay. Pooled mean values and the standard errors of two independent experiments (Exp. 2 ? 3) are shown in Fig. 5, and data from each experiment are given in Table 3. Homogeneous irradiation (HF) reduced cell viability to 33 ± 3 % compared to unirradiated controls, while microchannel irradiation resulted in 85 ± 10 % cell viability. The widened channels lead to tissue viabilities of 75 ± 6 % (WC) and 74 ± 7 % (OC), respectively. The resulting values for homogeneous irradiation are significantly lower than all the three channel modes (P \ 0.05). The values of SC, WC and OC, however, were not significantly different using the unpaired t test.
WC, OC and HF), with three skin samples each in two separate experiments (Exp. 1 ? 2). The pooled data of the two independent experiments are given as mean values in the tables and shown in Fig. 6. The X-ray reference curve, obtained from irradiation with 200-kV X-rays, was already published in (Zlobinskaya et al. 2013). In order to quantify the genetic damage of the four irradiation modes compared to a homogeneous X-ray irradiation, a dose modification factor (DMF), similar to the RBE, was calculated, using the dose of the reference radiation 200-kV X-rays that produced a response equal to 2 Gy of protons. For the (nearly) homogeneous irradiation mode, the resulting pooled DMFHF was 1.21 ± 0.21. For microchannel irradiation, the resulting pooled DMFSC was 0.29 ± 0.11, and for the widened channels, DMFWC
Micronucleus test Tables 4 and 5 present the detailed results of micronucleus induction in binucleate keratinocytes after proton irradiation, determined at four different irradiation modes (SC,
Fig. 5 MTT test results. Cell viability of microchannel (SC), widened channel (WC), overlapping channel (OC) and homogeneous field (HF) irradiations was measured 10 day after irradiation relative to sham-treated, unirradiated controls (CO) by the MTT assay. Error bars represent the SD of the mean value of six independently irradiated EpiDermFTTM tissue samples, from two different experiments
Table 3 MTT test results of experiment 2 and 3
Fig. 4 Measured width of the resulting proton distributions at different distances behind the aluminum sheet. For the three independent experiments, Gaussian distributions at 4.5, 9, 19 and 38 mm behind the Al were analyzed with Gafchromic films
Viability (%)
Experiment 2
Experiment 3
Pooled
SC
72.9 ± 2.0
98.1 ± 1.9
85.5 ± 10.0
WC
67.8 ± 1.4
75.6 ± 1.5
74.6 ± 5.1
OC
64.3 ± 2.2
81.5 ± 1.9
74.2 ± 6.8
HF
33.4 ± 1.0
32.2 ± 1.9
32.8 ± 2.9
Table 2 Measured width r for the widened channels in the three independent experiments r (lm)
Experiment 1
Experiment 2
Experiment 3
Mean ± SD
WC
267 ± 7
261 ± 7
247 ± 7
258 ± 11
OC
520 ± 15
520 ± 15
503 ± 15
514 ± 10
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Radiat Environ Biophys (2015) 54:335–342
Table 4 Frequency and distribution of micronuclei (MN) in cytokinesis-blocked (CB) binucleated epidermal keratinocytes from skin tissue induced by 2 Gy of 20 MeV protons at four spatial irradiation modes ID number
Group
Analyzed cells
Micronuclei per CB cell y (±SE)
Intercellular distribution of micronuclei
Dispersion ratio
0
r2/y
1
2
3
1
HF
465
0.075 ± 0.017
441
15
7
2
1.67
2
HF
483
0.093 ± 0.019
455
14
11
3
1.80
3 P G1–G3
HF HF
505 1453
0.050 ± 0.013 0.072 ± 0.009
489 1385
9 38
5 23
2 7
1.79 1.77
4
SC
440
0.025 ± 0.011
434
2
3
1
2.07
5
SC
493
0.034 ± 0.012
483
5
3
2
2.03
6 P G4–G6
SC
503
0.026 ± 0.008
492
9
2
0
1.28
SC
1436
0.029 ± 0.006
1409
16
8
3
1.80
7
WC
500
0.046 ± 0.012
484
10
5
1
1.65
8
WC
496
0.040 ± 0.012
483
8
3
2
1.86
9 P 7–9
WC
466
0.054 ± 0.015
450
10
3
3
1.91
WC
1462
0.047 ± 0.008
1417
28
11
6
1.81
10
OC
510
0.049 ± 0.013
493
10
6
1
1.67
11
OC
459
0.046 ± 0.015
448
5
2
4
2.29
12 P 10–12
OC
429
0.049 ± 0.013
413
12
3
1
1.53
OC
1398
0.048 ± 0.008
1354
27
11
10
1.82
Results from experiment 1 Table 5 Frequency and distribution of micronuclei (MN) in cytokinesis-blocked (CB) binucleated epidermal keratinocytes from skin tissue induced by 2 Gy of 20 MeV protons at four spatial irradiation modes ID number
Group
Analyzed cells
Micronuclei per CB cell y (±SE)
Intercellular distribution of micronuclei
Dispersion ratio
0
1
2
3
r2/y
1
HF
508
0.075 ± 0.016
484
11
12
1
1.718
2
HF
518
0.070 ± 0.015
492
17
8
1
1.545
3 P G1–G3
HF HF
495 1521
0.061 ± 0.016 0.068 ± 0.009
477 1453
10 38
4 24
4 6
2.010 1.740
4
SC
510
0.026 ± 0.008
498
11
1
0
1.130
5
SC
460
0.013 ± 0.007
456
2
2
0
1.663
6 P G4–G6
SC
502
0.028 ± 0.011
493
6
1
2
1.975
SC
1472
0.022 ± 0.005
1447
19
4
2
1.586
7
WC
509
0.024 ± 0.009
500
7
1
1
1.645
8
WC
298
0.057 ± 0.017
285
9
4
0
1.417
9 P 7–9
WC
502
0.038 ± 0.011
486
14
1
1
1.386
WC
1309
0.037 ± 0.007
1271
30
6
2
1.463
10
OC
502
0.054 ± 0.014
484
11
5
2
1.764
11
OC
517
0.070 ± 0.015
491
17
8
1
1.545
12 P 10–12
OC
516
0.043 ± 0.013
502
8
4
2
1.872
OC
1535
0.055 ± 0.008
1477
36
17
5
1.698
Results from experiment 2
was 0.64 ± 0.14 and DMFOC was 0.93 ± 0.17. The micronucleus frequency was significantly reduced compared to the homogeneous irradiation for the microchannel mode (SC) and the widened channels (WC) (P \ 0.01), but not for the overlapping channels (OC) (P [ 0.05).
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Discussion The aim of this study was to follow up on our pilot study on proton microchannel radiotherapy (Zlobinskaya et al. 2013) with regard to the so-called intermediate region
Radiat Environ Biophys (2015) 54:335–342
Fig. 6 Micronucleus (MN) induction in cytokinesis-blocked (CB) binucleate keratinocytes from EpiDermFTTM tissues. Linear dose– response curve for 200-kV X-rays (open circles, cf. Zlobinskaya et al. 2013) and mean MN frequency for homogeneous (HF) and microchannel irradiation modes (SC, WC, OC). Uncertainty bars represent the SD of the mean value of six tissue samples, from two different experiments
between the skin and the tumor, where the microchannels widen with increasing depth in tissue, resulting in an inhomogeneous dose distribution. This is an important step for a complete evaluation of the potential of proton microchannel irradiations for radiotherapy, after the benefits of this method for normal tissue have been demonstrated earlier (Zlobinskaya et al. 2013). The increasing channel size with depth is inherent for microchannel particle therapy due to small-angle scattering effects and is used to obtain a homogeneous dose distribution inside the tumor. It is important to note that widening of the channels is smaller for heavier ions than protons, but might be enhanced for all kinds of ions by an additional beam divergence. This widening has never been investigated in the context of X-ray microbeam radiation therapy (MRT), where arrays of microscopically thin and nearly parallel synchrotrongenerated X-ray beams are used (Serduc et al. 2008; Laissue et al. 2007; Brauer-Krisch et al. 2003, 2005). Typical sizes of the microplanar beams in MRT are 25–75 lm, separated by distances of 50–400 lm. This radiotherapy method, which has been examined in several animal studies (Anschel et al. 2010), maintains the microarray/channel geometry within the tumor as X-ray beams barely spread in depth and has been shown to prevent normal tissue necrosis, especially of the brain through sparing of normal brain vessels, while efficiently ensuring tumor control. In this study, the acute radiation effects and the induced genetic damage have been investigated for beam sizes that would be obtained in the deeper lying tissues when applying microchannel proton therapy. The irradiation effects
341
in the well-established human skin model (EpiDermFTTM, EFT400, MatTek) are compared for the different beam sizes and a homogeneous proton irradiation, and the experiments may serve as a representative for normal tissue effects. The comparative analysis shows no disadvantages of microchannel radiotherapy compared to conventional homogeneous broad-beam irradiations in the intermediate region, but argues for microchannel irradiation with regard to tissue viability and genetic damage even there. Cytotoxicity could be decreased by about a factor of two for all sizes of microchannels compared to homogeneous irradiation. Even though an increasing number of micronuclei per divided cell, corresponding to higher radiation damage, is induced with increasing channel size, there is still a significant reduction of genetic damage for the widened channels (as in 1/3 of the tumor depth) compared to the homogeneous irradiation. Also for the almost homogeneously overlapping channels (as in 2/3 of the tumor depth), the genetic damage does not exceed that of a homogeneous irradiation. These findings support and confirm the conclusion of our first study (Zlobinskaya et al. 2013) that proton microchannel radiotherapy leads to reduced acute and genotoxic effects compared to conventional broad-beam irradiation but keeping the homogeneous dose distribution inside the tumor and thus the tumor control as in conventional tumor therapy. Therefore, it could be an option in clinical proton and/or heavy ion therapy that would allow reduction of side effects keeping the same tumor control or enhancing tumor dose and thus improving tumor control in radiation resistant tumor tissues at still acceptable side effects. Acknowledgments This work was supported by the DFG Cluster of Excellence ‘‘Munich-Centre for Advanced Photonics,’’ by EU FP7 DoReMi Network of Excellence and by the Maier Leibnitz Laboratory Munich. We thank Sabine Reinhardt for her support with the Gafchromic film dosimetry and the staff of MLL operating the tandem accelerator.
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