International Journal of Radiation Biology, 2014; 90: 248–255 © 2014 Informa UK, Ltd. ISSN 0955-3002 print / ISSN 1362-3095 online DOI: 10.3109/09553002.2014.874608
Abscopal effect of radiation therapy: Interplay between radiation dose and p53 status
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Lidia Strigari1, Mariateresa Mancuso2, Valentina Ubertini4, Antonella Soriani1, Paola Giardullo3, Marcello Benassi1,5, Daniela D’Alessio1, Simona Leonardi2, Silvia Soddu4 & Gianluca Bossi4 1Laboratory of Medical Physics and Expert Systems, Regina Elena National Cancer Institute, Rome, 2Laboratory of Radiation
Biology and Biomedicine, ENEA CR-Casaccia, Rome, 3Department of Radiation Physics, Guglielmo Marconi University, Rome, 4Department of Experimental Oncology, Regina Elena National Cancer Institute, Rome, Italy, and 5Service of Medical Physics, Scientific Institute of Tumours of Romagna I.R.S.T., Meldola, Italy The abscopal effect refers to distant/non-targeted effects that were first described by Mole (1953) and have been sporadically documented since then. Although a variety of underlying biologic events have been hypothesized, this effect seems to be generated by multiple mechanisms and is still being investigated. It should be stressed that abscopal effects are not bystander effects in the traditional sense (Kaminski et al. 2005) but refer to radiation responses in areas separate from the irradiated tissue and presumably mediated by secreted soluble factors (Ohba et al. 1998). More recently, there has been a tendency to use the term bystander to describe the indirect radiation induced effect on non-irradiated tumour cells. In the treatment of many solid tumours, RT has been empirically combined with surgery in an attempt to increase local tumour control (LC) in respect to that obtained with a single modality (Cividalli et al. 2005). Recently, due to the diffusion of dedicated mobile linear accelerators, irradiation of the tumour bed during surgical procedures has become more feasible. The aim is to prevent clonogenic tumour cell proliferation which may represent the main obstacle to tumour radio-curability, particularly in fast repopulating cancers, such as head-and-neck tumours. Intra-Operative-RT (IORT) has been proposed as a post-operative adjuvant-therapy (ADJ), given the importance of the time interval after surgery for residual cell repopulation. In particular, IORT is essential when surgery is not fully effective in terms of LC, due to microscopic infiltration of subclinical residual cells in the tumour bed (Cividalli et al. 2005, Creton et al. 2006, Strigari et al. 2008). IORT, which enables the displacement of normal tissues, has the potential to improve LC in advanced tumours while sparing organs at risk. Moreover, the use of IORT or radio-surgical approaches to improve LC in intra and extra-cranial tumours have been supported by the advantage gained from more biologically effective, single, high doses on the tumour bed. In addition, when mobile accelerators are placed in unshielded operating rooms, the protection of
Abstract Purpose: This study investigates whether the abscopal effect induced by radiation-therapy (RT) is able to sterilize non-irradiated tumour cells through bystander signals. Material and methods: Wild-type (wt)-p53 or p53-null HCT116 human colon cancer cells were xenografted into both flanks of athymic female nude mice. When tumours reached a volume of 0.2 cm3, irradiation was performed, under strict dose monitoring, with a dedicated mobile accelerator designed for intra-Operative-RT (IORT). A dose of 10 or 20 Gy (IR groups), delivered by a 10 MeV electron beam, was delivered to a tumour established in one side flank, leaving the other non-irradiated (NIR groups). A subset of mice were sacrificed early on to carry out short-term molecular analyses. Results: All directly-irradiated tumours, showed a dose-dependent delayed and reduced regrowth, independent of the p53 status. Importantly, a significant effect on tumour-growth inhibition was also demonstrated in NIR wt-p53 tumours in the 20 Gy-irradiation group, with a moderate effect also evident after 10 Gy-irradiation. In contrast, no significant difference was observed in the NIR p53null tumours, independent of the dose delivered. Molecular analyses indicate that p53-dependent signals might be responsible for the abscopal effect in our model system, via a pro-apoptotic pathway. Conclusions: We suggest that the interplay between delivered dose and p53 status might help to sterilize out-of-field tumour cells. Keywords: IORT, in vivo abscopal effect, tumour growth, apoptosis, p53
Introduction The indirect anticancer effect of radiation therapy (RT) on tumour cells outside the irradiation field has been referred to as an abscopal/bystander effect in many human malignancies.
Correspondence: Lidia Strigari, Laboratory of Medical Physics and Expert System, Regina Elena National Cancer Institute Via E. Chianesi 53, 00144 Rome, Italy. Tel: ⫹ 39 6 5266 5602. Fax: ⫹ 39 6 5266 2740. E-mail: [email protected] (Received 17 April 2013; revised 27 August 2013; accepted 26 November 2013)
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Abscopal effect in a pre-clinical model of radiotherapy 249 the operators needs to be investigated (Strigari et al. 2004, Jaradat and Biggs 2008, Soriani et al. 2010). It is well established that the altered metabolism exhibited by cancer cells, including high rates of glycolysis, lactate production, biosynthesis of lipids and nucleotides, and the altered immune response, play an essential role in cancer progression (Demaria et al. 2004, Shen et al. 2012). Recently, the tumour suppressor gene TP53 was found to play a central role in this process. This gene is the most common target for genetic alteration in human tumours: More than 50% of all human cancers carry mutations within the p53 locus (Hainaut and Hollstein 2000, Petitjean et al. 2007). It is intriguing that the p53 status can modulate abscopal effects in vivo, such as tumour reduction caused by irradiating healthy mouse tissues (Camphausen et al. 2003). Due to the fact that in the clinical setting, we irradiate the residual tumour cells which remain in the tumour bed after surgery, we asked whether the presence/absence of p53 could help to sterilize non-irradiated tumour cells via bystander signals. Thus we set out to analyze in vivo the correlation between the p53 status of tumour cells and the radiation dose able to achieve LC.
Materials and methods
section. After irradiation, performed at the same time for each experiment, tumour-growth was monitored by calliper measurements twice a week, and tumour volumes [TV(cm3)] estimated by the formula: TV ⫽ (a)2 ⫻ b/2, where a and b are tumour width and length, respectively. Animals were weighed weekly to assess any effects consequent to irradiation, and no significant effects were observed with respect to the non-irradiated CT groups. At specific time points the animals were euthanized, the tumours excised, and fixed as follows: 10% PBS buffered formalin (24 h); 50% ethanol (24 h); 70% ethanol until immunohistochemical analysis. All procedures involving animals and their care were conducted in accordance with institutional guidelines and regulations and the animals were housed and kept in our facility.
Irradiation and dose verification Irradiation was delivered with a mobile accelerator Light Intraoperative Accelerator (Liac®12MeV; SORDINA SpA, Italy) specifically designed to perform IORT with four clinical (6, 8, 10 and 12 MeV) electron beam energies in operating theatres. At 10 MeV, R50 value (i.e., the value of depth in water at which the dose is 50% of its maximal value) is 38 mm. The dose was delivered using the 10 MeV energy, an applicator diameter of 30 mm. The delivery time of 10 and 20 Gy was 20 and 40 sec, respectively. Irradiation was performed within 1 h for all experimental groups.
Cell lines Human colon cancer (HCC) cells HCT116, bearing wild-type (wt)-p53 or p53-null (kindly provided by Dr Fanciulli, Regina Elena Cancer Institute), were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Eurobio, Les Ulis, France), 10% Fetal Bovine Serum (FBS; GIBCO-BRL, Grand Island, NY, USA), 2.0 mML-glutamine (Life Technologies Inc., Eggenstein, Germany), 100 U/ml Penicillin-100 μg/ml Streptomycin (Life Technologies Inc.). All cell lines were maintained at 37°C in a humidified environment of 5% CO2.
Irradiation set-up
Animals, tumour system, and treatments
We performed in vivo dosimetry (IVD) at a specific point using Metal Oxide Silicon Field Effect Transistors (i.e., MOSFET, Best Medical Canada, Ottawa, Ontario) appropriately calibrated as previously reported (Soriani et al. 2007). Dose verification in a plan under the mouse was performed by using MD-V2-55 (International Specialty Product, Wayne, NJ, USA) and EBT2 Gafchromic film (International Specialty Product, Wayne, NJ, USA) appropriately calibrated as reported in the Supplementary calibration data (available online at http://informahealthcare.com/doi/ abs/ 10.3109/09553002.2014.874608). Both MD-V2-55 and EBT2 Gafchromic films were cut in pieces of 4 ⫻ 8 cm2 and placed under the mice during irradiation (Figure 1b and c).
All experiments were performed in 40-day old athymic female nude mice (nu/nu CD1, Charles River Laboratories, Italy). Xenograft tumours were generated as previously reported (Bossi et al. 2008). Exponentially growing wt-p53 (4.0 ⫻ 106/flank side) or p53-null (2.0 ⫻ 106/flank side) HCT116 cells were subcutaneously injected into the right and left flanks of each animal. The number of cells to be injected was experimentally determined in order to obtain a comparable tumour volume (0.1–0.2 cm3) at the time of irradiation, about 3–4 weeks post-injection (Figure 1a). For each experiment, wt-p53 or p53-null HCT116 tumour-bearing mice were divided into three subgroups (n ⫽ 10 mice/group) as follows: (i) Mice with both tumours non-irradiated (CT), (ii) mice with one tumour irradiated with 10 Gy (IR 10 Gy), and one non-irradiated (NIR 10 Gy); (iii) mice with one tumour irradiated with 20 Gy (IR 20 Gy) and one nonirradiated (NIR 20 Gy). Before irradiation mice were anesthetized by intramuscular injection of 25 mg/kg Zoletil 50 (Virbac, LLDD-BP27-06511 Carros Cedex, France). A dose of 10 or 20 Gy was delivered as described in the next
Mice were placed in a polymethyl methacrylate (PMMA) box above a bolus. The xenograft tumour was located away from organs at risk and kept fixed in the irradiation field by using the automatic movements of LIAC allowed by the robotic system (Figure 2). In addition, the mice were shielded using a 5 mm PMMA plate and 2 mm of lead. The scattered dose was assessed using Gafchromic film, as described below.
Dose verification
Histology and immunohistochemical analyses A subset of animals from each experimental group (n ⫽ 2) were sacrificed 4 h post-irradiation; tumours were collected and processed for histological analysis, using standard methods and after staining with Hematoxylin & Eosin (H&E) the sections were analyzed.
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Figure 1. Experimental design. (a) In vivo set-up of the injected number of cells with null-or wt-p53. Points are the mean tumour size based on n ⫽ 4 individual tumours. In brackets the percentage of tumour uptake is reported. (b) Mice placed in a PMMA box above a bolus; the tumour is located away from the organs at risk and is kept fixed in the irradiation field using the LIAC applicator. The mouse is shielded using a 5 mm PMMA plate and 2 mm of lead. (c) Beam eye view of the mouse, LIAC applicator and gafchromic film.
For immunohistochemical analyses, 3-μm thick tumour sections were de-waxed, rehydrated then incubated in 0.3% H2O2 in methanol for 30 min to inhibit endogenous peroxidase. After antigen unmasking, carried out according to the manufacturer ’s instructions, sections were incubated overnight at ⫹ 4°C with the following primary antibodies: Anti-p53 (monoclonal, 1:25; DO7 Dako Cytomation, Glostrup, Denmark, kindly provided by Dr Mottolese, Regina Elena Cancer Institute); anti-BCL2associated X protein (Bax; polyclonal, 1:50; Cell Signaling Technology, Beverly, MA, USA); anti-cleaved caspase-3 (polyclonal, 1:100; Cell Signaling Technology). Detection of p53 and caspase-3 cleavage was carried out with the Vectastain Elite ABC Kit (Vector Laboratories, Inc., Burlingame, CA, USA), by using mouse or rabbit biotinylated secondary antibodies respectively, for 1 h at room temperature. After incubation with avidin-biotin immunoperoxidase, immunohistochemical staining was visualized with 3-amino-9-ethylcarbazole (Vector Novared kit; Vector Laboratories, Inc.). Detection of the Bax signal was performed using a horseradish peroxidase-conjugated
secondary antibody and the 3,3¢ Diaminobenzidine (DAB) chromogen system (Dako North America, Inc, Carpinteria, CA, USA). Quantification of caspase-3 positive cells was carried out collecting four digital images per tumour section (IAS imageprocessing software, Delta Sistemi, Rome, Italy). In order to standardize the sampling, images were collected from peripheral tumour areas, normally characterized by higher immunoreactivity. Total positive and negative cells were counted using a double-blind method.
Power calculation – statistical analysis Assuming a difference of at least 20%, a sample size of eight mice was considered appropriate to obtain an α ⫽ 0.05 and a statistical power of 80%. The percentage of tumour regrowth relative to the volume at the time of irradiation was calculated at week 8/9 post-irradiation and compared with each group using the Fisher test. Apoptotic indexes are reported as total number of caspase-3-positive vs. negative cells, and the Chi square-test was used. Analyses were performed using the R-Software (URL http://www.R-project.org) or GraphPad
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Abscopal effect in a pre-clinical model of radiotherapy 251
Figure 2. Gafchromic records and dose distribution. (a) Six irradiated gafchromic film pieces. Tumours are visible at the edge of the irradiated film with a circular applicator (30 mm diameter). Dose distribution (cGy) along a line across the tumour (along the applicator diameter) for the dose of 10 (b) and 20 Gy (c), respectively.
Prism vers.5 for Windows (GraphPad Software, San Diego, CA, USA). P-values ⱕ 0.05 were considered statistically significant.
Results
achieve a similar tumour-growth rate in our panel of cancer lines we performed primary experiments in vivo. As reported in Figure 1a, tumours derived from wt-p53 and p53-null HCT116 cells had a similar growth rate when transplanted at 4.0 ⫻ 106 and 2.0 ⫻ 106/flank side, respectively, with 100% tumour uptake.
Experimental tumour model Two well-characterized wt-p53 and p53-null HCT116 cancer cell lines (Mothersill et al. 2011) were used in the present study. To identify the experimental conditions required to
Dosimetric data Figure 1b and c show the mouse irradiation, and the measurement set-up immediately after irradiation and removal of
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Treatment and tumour response To investigate whether the abscopal effect is dependent on the p53 status and delivered dose, tumour-growth delay studies were performed in wt-p53 and p53-null HCT116 xenograft tumours generated in both flanks of nude mice, following optimized experimental conditions. RT treatment was performed on established tumours (approximately 0.2 cm3), one lesion/mouse, by delivering 10 or 20 Gy doses, and the effects on IR and NIR tumour-growth followed by calliper measurements twice a week. As reported in Fig-
(a)
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ure 3, all IR tumours, independent of p53-status, showed a dose dependent tumour-growth inhibition in respect to their CT counterparts (Figure 3b). Of importance, the NIR wt-p53 tumours showed a significant tumour-growth inhibition in the 20 Gy-irradiation group and only a moderate effect in the NIR 10 Gy-irradiation group (Figure 3b). Interestingly, the tumour-growth inhibition observed on NIR wt-p53 tumours upon 20 Gy irradiation was comparable to that observed in IR wt-p53 tumours after 10 Gy irradiation (Figure 3b). In contrast, NIR p53-null tumours showed no significant delay in tumour-growth with respect to the p53null CT group, independent of delivered dose (20 Gy or 10 Gy) (Figure 3a). Figure 4 shows a representative picture of null- (Figure 4a) and wt-p53 (Figure 4b) tumour-bearing mice at week 8/9 post-irradiation, respectively (arrows indicate the IR lesions). Overall, our results show that the identified experimental model is suitable for performing studies on the abscopal effect in vivo, and suggest that the p53 status and high dose delivered may promote an in vivo abscopal effect.
Short-term molecular analyses To better characterize the RT response in our experimental model, and understand the molecular mechanisms involved in tumour-growth inhibition, we analyzed the apoptotic pathway in a subset of tumours/group 4 h post-irradiation.
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the shielding system. The images of six irradiated film pieces with visible tumour burden are shown in Figure 2a. The dose distribution in cGy along a line across the tumour (along the diameter of the applicator) for the doses of 10 and 20 Gy is also shown (Figure 2b and c). A correlation between MOSFET and MD-V2-55 was observed with Pearson coefficients of 0.95 and 0.98 for measurement at the gafchromic surface or under tumours, respectively (p ⬍ 0.0001). Both MD-V2-55 and EBT2 indicated an out-of-field dose lower than 50 cGy close to the PMMA applicator and lower than 10 cGy at a distance of 3 cm from the applicator, with a maximal dose of 50 cGy to the NIR (under the lead and PMMA shield).
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Figure 3. High dose delivery and wt-p53 contribute to the bystander effects in vivo. Tumour-growth of IR and NIR tumours; the control non-irradiated mice are also reported. (a) p53-null; (b) wt-p53. Volumetric data were obtained using 8 mice/group. The difference between mean volume ratios of directly irradiated (10 or 20 Gy) tumours and controls was statistically significant, as well as the difference between tumours shielded while the other tumours received a dose of 20 Gy and controls. Representative results of two independent experiments are reported.
Figure 4. High dose delivery and wt-p53 contribute to the abscopal/ bystander effect in vivo. Mice carrying null- (a) or wt-p53 (b) tumours at 8/9 weeks post-irradiation respectively. IR lesions are indicated by arrows. Of note, the total tumour burden is less than 10% of the total body weight, as allowed by our animal use guidelines.
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Abscopal effect in a pre-clinical model of radiotherapy 253
Figure 5. Molecular analyses in wt-p53 tumours 4 h post-irradiation. Representative images of immunohistochemical staining carried out using antibodies directed against the pro-apoptotic factor Bax (left column), cleaved caspase-3 (middle column) and p53 (right column). (a) Quantification of cleaved caspase-3 positive cells in wt-p53 tumours. Representative results of two independent experiments are reported.
Results relative to wt-p53 tumours are shown in Figure 5. Immunohistochemistry carried out using an antibody directed against the pro-apoptotic factor Bax (Figure 5, left column), shows that none of the tumours from the CT group showed Bax expression, whereas, in both IR and NIR wt-p53 tumours a high immunoreactivity was revealed with both of the RT-doses, resulting in a clear dose-dependent response. To better characterize the apoptotic pathway, we then performed immunohistochemical staining to assess caspase-3 cleavage, the main executioner of apoptosis (Figure 5, middle column). Quantification of caspase-3 positive cells was performed for all irradiation settings as shown in Figure 5a. Similar to the Bax expression, IR wt-p53 tumours showed a statistically significant difference in the number of caspase-3 positive cells when compared to the CT group (p ⬍ 0.0001). The 20 Gy dose induced a higher apoptotic response when compared to the 10 Gy irradiation group (p ⫽ 0.0331). Of note, a clear dose-dependence for apoptosis was also found in the NIR wt-p53 tumours (20 Gy NIR vs.10 Gy NIR, p ⬍ 0.0001). Apoptotic values in these tumours were also statistically significant when compared to the CT group (p ⬍ 0.0001). Short-term molecular analyses relative to p53-null tumours are shown in Figure 6. Although IR p53-null tumours showed a strong Bax expression, no staining was
observed in NIR p53-null tumours, independent of the dose delivered, similar to CT tumours. After the quantification of caspase-3 positive cells (Figure 6a), IR p53-null tumours showed a significant apoptotic response albeit attenuated with respect to IR wt-p53 tumours. In spite of the lower caspase-3 activation, apoptotic values were statistically significant when compared to their CT for both delivered doses (p ⬍ 0.0001). In contrast, no significant apoptotic response was observed in NIR p53-null tumours after a delivered dose of 10 or 20 Gy. Of interest, values of caspase-3 positive cells were always found to be statistically significant when IR vs. NIR were compared in wt-p53 as well as p53-null tumours. Conversely, similar analysis performed to explore the modulation of the anti-apoptotic bcl-2 molecules in the IR and NIR tumours showed no significant effects in our experimental model (data not shown). The overall results indicate that, although both p53independent and -dependent apoptotic pathways are involved in IR tumour-growth inhibition, the markedly higher caspase-3 activation shown by NIR wt-p53 tumours suggests that the radiation-induced abscopal effect in vivo is strongly dependent on the functional p53 status. In line with this, immunohistochemical staining performed to detect p53 expression showed that p53 positivity increased with the dose in cancer tissues, but was lacking in wt-p53 CT as well as
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Figure 6. Molecular analyses in p53-null tumours 4 h post-irradiation. Representative images of immunostaining with Bax (left column), cleaved caspase-3 (middle column) and p53 (right column). (a) Quantification of cleaved caspase-3 positive cells in p53-null tumours. Representative results of two independent experiments are reported.
the p53-null tumour groups (Figures 5 and 6, right columns). However of relevance, in the NIR wt-p53 tumours the p53 expression was found to be significantly higher upon 20 Gy irradiation while with 10 Gy it was only slightly higher.
Discussion According to the IARC database, 50–70% of human cancers express the wt-p53 gene, depending on the tumour histotype. A plethora of data now available concerning the bystander effect fall into two separate experimental categories, and there is some doubt whether the two groups of experiments are addressing the same phenomenon. First, in vitro experiments involving the transfer of conditioned medium from irradiated wt-p53 and p53-null HCT116 cells showed biologic effects only in non-irradiated wt-p53 cells, suggesting that bystander signals are produced by both wt-p53 and p53-null cells, however only wt-p53 cells respond to them (Mothersill et al. 2011). Secondly, the use of single particle micro beams allows irradiation of specific cells, and thus the study of the biological effects in neighbouring cells. At present, however, only a few number of studies dedicated to explore the abscopal/bystander effect in vivo have been reported. Studies of tumour induction have shown the oncogenic properties of
bystander effects in Patched heterozygous mouse cerebellum (Mancuso et al. 2008). Other studies, have reported the involvement of wt-p53 in the radiation induced bystander antitumour effects, showing NIR tumour-growth delay in wtp53 but not p53-null tumour-bearing mice after irradiation of normal tissue (i.e., right hind leg) (Camphausen et al. 2003). Recently, other studies have shown that combined RT and CTLA-4 neutrilizing antibody promotes radiation induced bystander antitumour effects in NIR tumours (Dewan et al. 2009). Currently, data aimed at assessing the possible role of wt-p53 in radiation-induced bystander effects are contradictory and need to be investigated further. In the present study, through an in vivo experimental approach co-implanting either wt-p53 or p53-null HCT116 cells in both flanks of nude mice and verifying the tumour-growth delay effects upon RT, we were able to observe bystander effects in NIR wt-p53 tumours with a single high dose of 20 Gy delivered to the contra-lateral lesion, demonstrating the relevance of delivered total dose to IR tumours. The results were confirmed also with a different wt-p53 carrying tumour line (data not shown). At the molecular level, short-term analyses, performed on tumours at 4 h postirradiation, showed markedly higher caspase-3 activation in wt-p53 NIR tumours, suggesting that the in vivo radia-
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Abscopal effect in a pre-clinical model of radiotherapy 255 tion-induced bystander effect is principally dependent on the functional p53 status. Conversely, both p53-independent and -dependent apoptotic pathways are involved in IR tumour-growth inhibition. Notably, this study is the first at investigating the effects upon single dose delivery with IORT, where 10 and 20 Gy can be considered equivalent to 20 and 50 Gy delivered at 2 Gy/fraction (conventional fractionation), respectively, assuming an α/β ratio of 10 Gy. Conclusively, our results suggest that the interplay between radiation dose and p53 status plays a critical role in the RT-induced bystander effects (Figure 5). To better assess whether functional p53 protein is required for production of RT-induced abscopal signals in IR cells or bystander effects in NIR cells, similar tumor growth delay studies were performed by co-implanting wt-p53 and null-p53 cancer cells in each flank-side of nude mice and delivering single RT dose (20 Gy) to either wt-p53 or null-p53 lesion. Results, in agreement with in vitro studies (Mothersill et al. 2011), have shown that both IR null- and wt-p53 tumors produce abscopal signals, but functional p53 protein is required to produce bystander effects in NIR lesion (data not shown). Our results are intriguing and could have a wide impact on tailored radiotherapy based on p53 genetic alteration by helping to sterilize nonirradiated tumour cells through bystander signals. In fact, the abscopal effect could be advantageous where the failure after high total doses, such as those delivered in current therapy, in high-risk, i.e., breast or prostate cancer, patients is mainly due to distant disease progression and not to local recurrence.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study was supported by Associazione per la Ricerca sul Cancro (AIRC) with grants to GB (IG#8804) and MM (IG#10357), and TOP IMPLART project (I11J10000420002).
References Bossi G, Marampon F, Maor-Aloni R, Zani B, Rotter V, Oren M, Strano S, Blandino G, Sacchi A . 2008. Conditional RNA interference in vivo to study mutant p53 oncogenic gain of function on tumour malignancy. Cell Cycle 7:1870–1879. Camphausen K, Moses MA , Ménard C, Sproull M, Beecken WD, Folkman J, O’Reilly MS. 2003. Radiation abscopal antitumour effect is mediated through p53. Cancer Res 63:1990–1993. Cividalli A, Creton G, Ceciarelli F, Strigari L, Tirindelli Danesi D, Benassi M. 2005. Influence of time interval between surgery and
Supplementary material available online Supplementary calibration data
radiotherapy on tumour regrowth. J Experim Clin Cancer Res 24: 109–116. Creton G, Benassi M, Di Staso M, Ingrosso G, Giubilei C, Strigari L. 2006. The time factor in oncology: Consequences on tumour volume and therapeutic planning. J Experim Clin Cancer Res 25:557–573. Demaria S, Ng B, Devitt ML, Babb JS, Kawashima N, Liebes L, Formenti SC. 2004. Ionizing radiation inhibition of distant untreated tumours (abscopal effect) is immune mediated. Int J Radiat Oncol Biol Phys 58:862–870. Dewan MZ, Galloway AE, Kawashima N, Dewyngaert JK , Babb JS, Formenti SC, Demaria S. 2009. Fractionated but not single-dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody. Clin Cancer Res 15: 5379–5388. Hainaut P, Hollstein M. 2000. p53 and human cancer: The first ten thousand mutations. Adv in Cancer Res 77:81–137. Jaradat AK , Biggs PJ. 2008. Measurement of the neutron leakage from a dedicated intraoperative radiation therapy electron linear accelerator and a conventional linear accelerator for 9, 12, 15(16), and 18(20) MeV electron energies. Medical Phys 35:1711–1717. Kaminski JM, Shinohara E, Summers JB, Niermann KJ, Morimoto A , Brousal J. 2005. The controversial abscopal effect. Cancer Treat Rev 31:159–172. Mancuso M, Pasquali E, Leonardi S, Tanori M, Rebessi S, Di Majo V, Pazzaglia S, Toni MP, Pimpinella M, Covelli V, Saran A . 2008. Oncogenic bystander radiation effects in Patched heterozygous mouse cerebellum. Proc Nat Acad Sci USA 105:12445–12450. Mole RH. 1953. Whole body irradiation: Radiobiology or medicine? Br J Radiobiol 26:234–241. Mothersill C, Bristow RG, Harding SM, Smith RW, Mersov A , Seymour CB. 2011. A role for p53 in the response of bystander cells to receipt of medium borne signals from irradiated cells. Int J Radiat Biol 87:1120–1125. Ohba K, Omagari K, Nakamura T, Ikuno N, Saeki S, Matsuo I, Kinoshita H, Masuda J, Hazama H, Sakamoto I, Kohno S. 1998. Abscopal regression of hepatocellular carcinoma after radiotherapy for bone metastasis. Gut 43:575–577. Petitjean A , Mathe E, Kato S, Ishioka C, Tavtigian SV, Hainaut P, Olivier M. 2007. Impact of mutant p53 functional properties on TP53 mutation patterns and tumour phenotype: Lessons from recent developments in the IARC TP53 database. Human Mutat 28: 622–629. Shen L, Sun X, Fu Z, Yang G, Li J, Yao L. 2012. The fundamental role of the p53 pathway in tumour metabolism and its implication in tumour therapy. Clin Cancer Res 18:1561–1567. Soriani A , Felici G, Fantini M, Paolucci M, Borla O, Evangelisti G, Benassi M, Strigari L. 2010. Radiation protection measurements around a 12 MeV mobile dedicated IORT accelerator. Med Phys 37:995–1003. Soriani A , Landoni V, Marzi S, Iaccarino G, Saracino B, Arcangeli G, Benassi M. 2007. Setup verification and in vivo dosimetry during intraoperative radiation therapy (IORT) for prostate cancer. Med Phys 34:3205–3210. Strigari L, D’Andrea M, Abate A , Benassi M. 2008. A heterogeneous dose distribution in simultaneous integrated boost: The role of the clonogenic cell density on the tumour control probability. Phys Med Biol 53:5257–5273. Strigari L, Soriani A , Landoni V, Teodoli S, Bruzzaniti V, Benassi M. 2004. Radiation exposure of personnel during intraoperative radiotherapy (IORT): Radiation protection aspects. J Experim Clin Cancer Res 23:489–494.
Abscopal effect of radiation therapy: Interplay between radiation dose and p53 status
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Lidia Strigari1, Mariateresa Mancuso2, Valentina Ubertini4, Antonella Soriani1, Paola Giardullo3, Marcello Benassi1,5, Daniela D’Alessio1, Simona Leonardi2, Silvia Soddu4 & Gianluca Bossi4 1Laboratory of Medical Physics and Expert Systems, Regina Elena National Cancer Institute, Rome, 2Laboratory of Radiation
Biology and Biomedicine, ENEA CR-Casaccia, Rome, 3Department of Radiation Physics, Guglielmo Marconi University, Rome, 4Department of Experimental Oncology, Regina Elena National Cancer Institute, Rome, Italy, and 5Service of Medical Physics, Scientific Institute of Tumours of Romagna I.R.S.T., Meldola, Italy The abscopal effect refers to distant/non-targeted effects that were first described by Mole (1953) and have been sporadically documented since then. Although a variety of underlying biologic events have been hypothesized, this effect seems to be generated by multiple mechanisms and is still being investigated. It should be stressed that abscopal effects are not bystander effects in the traditional sense (Kaminski et al. 2005) but refer to radiation responses in areas separate from the irradiated tissue and presumably mediated by secreted soluble factors (Ohba et al. 1998). More recently, there has been a tendency to use the term bystander to describe the indirect radiation induced effect on non-irradiated tumour cells. In the treatment of many solid tumours, RT has been empirically combined with surgery in an attempt to increase local tumour control (LC) in respect to that obtained with a single modality (Cividalli et al. 2005). Recently, due to the diffusion of dedicated mobile linear accelerators, irradiation of the tumour bed during surgical procedures has become more feasible. The aim is to prevent clonogenic tumour cell proliferation which may represent the main obstacle to tumour radio-curability, particularly in fast repopulating cancers, such as head-and-neck tumours. Intra-Operative-RT (IORT) has been proposed as a post-operative adjuvant-therapy (ADJ), given the importance of the time interval after surgery for residual cell repopulation. In particular, IORT is essential when surgery is not fully effective in terms of LC, due to microscopic infiltration of subclinical residual cells in the tumour bed (Cividalli et al. 2005, Creton et al. 2006, Strigari et al. 2008). IORT, which enables the displacement of normal tissues, has the potential to improve LC in advanced tumours while sparing organs at risk. Moreover, the use of IORT or radio-surgical approaches to improve LC in intra and extra-cranial tumours have been supported by the advantage gained from more biologically effective, single, high doses on the tumour bed. In addition, when mobile accelerators are placed in unshielded operating rooms, the protection of
Abstract Purpose: This study investigates whether the abscopal effect induced by radiation-therapy (RT) is able to sterilize non-irradiated tumour cells through bystander signals. Material and methods: Wild-type (wt)-p53 or p53-null HCT116 human colon cancer cells were xenografted into both flanks of athymic female nude mice. When tumours reached a volume of 0.2 cm3, irradiation was performed, under strict dose monitoring, with a dedicated mobile accelerator designed for intra-Operative-RT (IORT). A dose of 10 or 20 Gy (IR groups), delivered by a 10 MeV electron beam, was delivered to a tumour established in one side flank, leaving the other non-irradiated (NIR groups). A subset of mice were sacrificed early on to carry out short-term molecular analyses. Results: All directly-irradiated tumours, showed a dose-dependent delayed and reduced regrowth, independent of the p53 status. Importantly, a significant effect on tumour-growth inhibition was also demonstrated in NIR wt-p53 tumours in the 20 Gy-irradiation group, with a moderate effect also evident after 10 Gy-irradiation. In contrast, no significant difference was observed in the NIR p53null tumours, independent of the dose delivered. Molecular analyses indicate that p53-dependent signals might be responsible for the abscopal effect in our model system, via a pro-apoptotic pathway. Conclusions: We suggest that the interplay between delivered dose and p53 status might help to sterilize out-of-field tumour cells. Keywords: IORT, in vivo abscopal effect, tumour growth, apoptosis, p53
Introduction The indirect anticancer effect of radiation therapy (RT) on tumour cells outside the irradiation field has been referred to as an abscopal/bystander effect in many human malignancies.
Correspondence: Lidia Strigari, Laboratory of Medical Physics and Expert System, Regina Elena National Cancer Institute Via E. Chianesi 53, 00144 Rome, Italy. Tel: ⫹ 39 6 5266 5602. Fax: ⫹ 39 6 5266 2740. E-mail: [email protected] (Received 17 April 2013; revised 27 August 2013; accepted 26 November 2013)
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Abscopal effect in a pre-clinical model of radiotherapy 249 the operators needs to be investigated (Strigari et al. 2004, Jaradat and Biggs 2008, Soriani et al. 2010). It is well established that the altered metabolism exhibited by cancer cells, including high rates of glycolysis, lactate production, biosynthesis of lipids and nucleotides, and the altered immune response, play an essential role in cancer progression (Demaria et al. 2004, Shen et al. 2012). Recently, the tumour suppressor gene TP53 was found to play a central role in this process. This gene is the most common target for genetic alteration in human tumours: More than 50% of all human cancers carry mutations within the p53 locus (Hainaut and Hollstein 2000, Petitjean et al. 2007). It is intriguing that the p53 status can modulate abscopal effects in vivo, such as tumour reduction caused by irradiating healthy mouse tissues (Camphausen et al. 2003). Due to the fact that in the clinical setting, we irradiate the residual tumour cells which remain in the tumour bed after surgery, we asked whether the presence/absence of p53 could help to sterilize non-irradiated tumour cells via bystander signals. Thus we set out to analyze in vivo the correlation between the p53 status of tumour cells and the radiation dose able to achieve LC.
Materials and methods
section. After irradiation, performed at the same time for each experiment, tumour-growth was monitored by calliper measurements twice a week, and tumour volumes [TV(cm3)] estimated by the formula: TV ⫽ (a)2 ⫻ b/2, where a and b are tumour width and length, respectively. Animals were weighed weekly to assess any effects consequent to irradiation, and no significant effects were observed with respect to the non-irradiated CT groups. At specific time points the animals were euthanized, the tumours excised, and fixed as follows: 10% PBS buffered formalin (24 h); 50% ethanol (24 h); 70% ethanol until immunohistochemical analysis. All procedures involving animals and their care were conducted in accordance with institutional guidelines and regulations and the animals were housed and kept in our facility.
Irradiation and dose verification Irradiation was delivered with a mobile accelerator Light Intraoperative Accelerator (Liac®12MeV; SORDINA SpA, Italy) specifically designed to perform IORT with four clinical (6, 8, 10 and 12 MeV) electron beam energies in operating theatres. At 10 MeV, R50 value (i.e., the value of depth in water at which the dose is 50% of its maximal value) is 38 mm. The dose was delivered using the 10 MeV energy, an applicator diameter of 30 mm. The delivery time of 10 and 20 Gy was 20 and 40 sec, respectively. Irradiation was performed within 1 h for all experimental groups.
Cell lines Human colon cancer (HCC) cells HCT116, bearing wild-type (wt)-p53 or p53-null (kindly provided by Dr Fanciulli, Regina Elena Cancer Institute), were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Eurobio, Les Ulis, France), 10% Fetal Bovine Serum (FBS; GIBCO-BRL, Grand Island, NY, USA), 2.0 mML-glutamine (Life Technologies Inc., Eggenstein, Germany), 100 U/ml Penicillin-100 μg/ml Streptomycin (Life Technologies Inc.). All cell lines were maintained at 37°C in a humidified environment of 5% CO2.
Irradiation set-up
Animals, tumour system, and treatments
We performed in vivo dosimetry (IVD) at a specific point using Metal Oxide Silicon Field Effect Transistors (i.e., MOSFET, Best Medical Canada, Ottawa, Ontario) appropriately calibrated as previously reported (Soriani et al. 2007). Dose verification in a plan under the mouse was performed by using MD-V2-55 (International Specialty Product, Wayne, NJ, USA) and EBT2 Gafchromic film (International Specialty Product, Wayne, NJ, USA) appropriately calibrated as reported in the Supplementary calibration data (available online at http://informahealthcare.com/doi/ abs/ 10.3109/09553002.2014.874608). Both MD-V2-55 and EBT2 Gafchromic films were cut in pieces of 4 ⫻ 8 cm2 and placed under the mice during irradiation (Figure 1b and c).
All experiments were performed in 40-day old athymic female nude mice (nu/nu CD1, Charles River Laboratories, Italy). Xenograft tumours were generated as previously reported (Bossi et al. 2008). Exponentially growing wt-p53 (4.0 ⫻ 106/flank side) or p53-null (2.0 ⫻ 106/flank side) HCT116 cells were subcutaneously injected into the right and left flanks of each animal. The number of cells to be injected was experimentally determined in order to obtain a comparable tumour volume (0.1–0.2 cm3) at the time of irradiation, about 3–4 weeks post-injection (Figure 1a). For each experiment, wt-p53 or p53-null HCT116 tumour-bearing mice were divided into three subgroups (n ⫽ 10 mice/group) as follows: (i) Mice with both tumours non-irradiated (CT), (ii) mice with one tumour irradiated with 10 Gy (IR 10 Gy), and one non-irradiated (NIR 10 Gy); (iii) mice with one tumour irradiated with 20 Gy (IR 20 Gy) and one nonirradiated (NIR 20 Gy). Before irradiation mice were anesthetized by intramuscular injection of 25 mg/kg Zoletil 50 (Virbac, LLDD-BP27-06511 Carros Cedex, France). A dose of 10 or 20 Gy was delivered as described in the next
Mice were placed in a polymethyl methacrylate (PMMA) box above a bolus. The xenograft tumour was located away from organs at risk and kept fixed in the irradiation field by using the automatic movements of LIAC allowed by the robotic system (Figure 2). In addition, the mice were shielded using a 5 mm PMMA plate and 2 mm of lead. The scattered dose was assessed using Gafchromic film, as described below.
Dose verification
Histology and immunohistochemical analyses A subset of animals from each experimental group (n ⫽ 2) were sacrificed 4 h post-irradiation; tumours were collected and processed for histological analysis, using standard methods and after staining with Hematoxylin & Eosin (H&E) the sections were analyzed.
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Figure 1. Experimental design. (a) In vivo set-up of the injected number of cells with null-or wt-p53. Points are the mean tumour size based on n ⫽ 4 individual tumours. In brackets the percentage of tumour uptake is reported. (b) Mice placed in a PMMA box above a bolus; the tumour is located away from the organs at risk and is kept fixed in the irradiation field using the LIAC applicator. The mouse is shielded using a 5 mm PMMA plate and 2 mm of lead. (c) Beam eye view of the mouse, LIAC applicator and gafchromic film.
For immunohistochemical analyses, 3-μm thick tumour sections were de-waxed, rehydrated then incubated in 0.3% H2O2 in methanol for 30 min to inhibit endogenous peroxidase. After antigen unmasking, carried out according to the manufacturer ’s instructions, sections were incubated overnight at ⫹ 4°C with the following primary antibodies: Anti-p53 (monoclonal, 1:25; DO7 Dako Cytomation, Glostrup, Denmark, kindly provided by Dr Mottolese, Regina Elena Cancer Institute); anti-BCL2associated X protein (Bax; polyclonal, 1:50; Cell Signaling Technology, Beverly, MA, USA); anti-cleaved caspase-3 (polyclonal, 1:100; Cell Signaling Technology). Detection of p53 and caspase-3 cleavage was carried out with the Vectastain Elite ABC Kit (Vector Laboratories, Inc., Burlingame, CA, USA), by using mouse or rabbit biotinylated secondary antibodies respectively, for 1 h at room temperature. After incubation with avidin-biotin immunoperoxidase, immunohistochemical staining was visualized with 3-amino-9-ethylcarbazole (Vector Novared kit; Vector Laboratories, Inc.). Detection of the Bax signal was performed using a horseradish peroxidase-conjugated
secondary antibody and the 3,3¢ Diaminobenzidine (DAB) chromogen system (Dako North America, Inc, Carpinteria, CA, USA). Quantification of caspase-3 positive cells was carried out collecting four digital images per tumour section (IAS imageprocessing software, Delta Sistemi, Rome, Italy). In order to standardize the sampling, images were collected from peripheral tumour areas, normally characterized by higher immunoreactivity. Total positive and negative cells were counted using a double-blind method.
Power calculation – statistical analysis Assuming a difference of at least 20%, a sample size of eight mice was considered appropriate to obtain an α ⫽ 0.05 and a statistical power of 80%. The percentage of tumour regrowth relative to the volume at the time of irradiation was calculated at week 8/9 post-irradiation and compared with each group using the Fisher test. Apoptotic indexes are reported as total number of caspase-3-positive vs. negative cells, and the Chi square-test was used. Analyses were performed using the R-Software (URL http://www.R-project.org) or GraphPad
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Abscopal effect in a pre-clinical model of radiotherapy 251
Figure 2. Gafchromic records and dose distribution. (a) Six irradiated gafchromic film pieces. Tumours are visible at the edge of the irradiated film with a circular applicator (30 mm diameter). Dose distribution (cGy) along a line across the tumour (along the applicator diameter) for the dose of 10 (b) and 20 Gy (c), respectively.
Prism vers.5 for Windows (GraphPad Software, San Diego, CA, USA). P-values ⱕ 0.05 were considered statistically significant.
Results
achieve a similar tumour-growth rate in our panel of cancer lines we performed primary experiments in vivo. As reported in Figure 1a, tumours derived from wt-p53 and p53-null HCT116 cells had a similar growth rate when transplanted at 4.0 ⫻ 106 and 2.0 ⫻ 106/flank side, respectively, with 100% tumour uptake.
Experimental tumour model Two well-characterized wt-p53 and p53-null HCT116 cancer cell lines (Mothersill et al. 2011) were used in the present study. To identify the experimental conditions required to
Dosimetric data Figure 1b and c show the mouse irradiation, and the measurement set-up immediately after irradiation and removal of
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Treatment and tumour response To investigate whether the abscopal effect is dependent on the p53 status and delivered dose, tumour-growth delay studies were performed in wt-p53 and p53-null HCT116 xenograft tumours generated in both flanks of nude mice, following optimized experimental conditions. RT treatment was performed on established tumours (approximately 0.2 cm3), one lesion/mouse, by delivering 10 or 20 Gy doses, and the effects on IR and NIR tumour-growth followed by calliper measurements twice a week. As reported in Fig-
(a)
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ure 3, all IR tumours, independent of p53-status, showed a dose dependent tumour-growth inhibition in respect to their CT counterparts (Figure 3b). Of importance, the NIR wt-p53 tumours showed a significant tumour-growth inhibition in the 20 Gy-irradiation group and only a moderate effect in the NIR 10 Gy-irradiation group (Figure 3b). Interestingly, the tumour-growth inhibition observed on NIR wt-p53 tumours upon 20 Gy irradiation was comparable to that observed in IR wt-p53 tumours after 10 Gy irradiation (Figure 3b). In contrast, NIR p53-null tumours showed no significant delay in tumour-growth with respect to the p53null CT group, independent of delivered dose (20 Gy or 10 Gy) (Figure 3a). Figure 4 shows a representative picture of null- (Figure 4a) and wt-p53 (Figure 4b) tumour-bearing mice at week 8/9 post-irradiation, respectively (arrows indicate the IR lesions). Overall, our results show that the identified experimental model is suitable for performing studies on the abscopal effect in vivo, and suggest that the p53 status and high dose delivered may promote an in vivo abscopal effect.
Short-term molecular analyses To better characterize the RT response in our experimental model, and understand the molecular mechanisms involved in tumour-growth inhibition, we analyzed the apoptotic pathway in a subset of tumours/group 4 h post-irradiation.
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the shielding system. The images of six irradiated film pieces with visible tumour burden are shown in Figure 2a. The dose distribution in cGy along a line across the tumour (along the diameter of the applicator) for the doses of 10 and 20 Gy is also shown (Figure 2b and c). A correlation between MOSFET and MD-V2-55 was observed with Pearson coefficients of 0.95 and 0.98 for measurement at the gafchromic surface or under tumours, respectively (p ⬍ 0.0001). Both MD-V2-55 and EBT2 indicated an out-of-field dose lower than 50 cGy close to the PMMA applicator and lower than 10 cGy at a distance of 3 cm from the applicator, with a maximal dose of 50 cGy to the NIR (under the lead and PMMA shield).
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Figure 3. High dose delivery and wt-p53 contribute to the bystander effects in vivo. Tumour-growth of IR and NIR tumours; the control non-irradiated mice are also reported. (a) p53-null; (b) wt-p53. Volumetric data were obtained using 8 mice/group. The difference between mean volume ratios of directly irradiated (10 or 20 Gy) tumours and controls was statistically significant, as well as the difference between tumours shielded while the other tumours received a dose of 20 Gy and controls. Representative results of two independent experiments are reported.
Figure 4. High dose delivery and wt-p53 contribute to the abscopal/ bystander effect in vivo. Mice carrying null- (a) or wt-p53 (b) tumours at 8/9 weeks post-irradiation respectively. IR lesions are indicated by arrows. Of note, the total tumour burden is less than 10% of the total body weight, as allowed by our animal use guidelines.
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Abscopal effect in a pre-clinical model of radiotherapy 253
Figure 5. Molecular analyses in wt-p53 tumours 4 h post-irradiation. Representative images of immunohistochemical staining carried out using antibodies directed against the pro-apoptotic factor Bax (left column), cleaved caspase-3 (middle column) and p53 (right column). (a) Quantification of cleaved caspase-3 positive cells in wt-p53 tumours. Representative results of two independent experiments are reported.
Results relative to wt-p53 tumours are shown in Figure 5. Immunohistochemistry carried out using an antibody directed against the pro-apoptotic factor Bax (Figure 5, left column), shows that none of the tumours from the CT group showed Bax expression, whereas, in both IR and NIR wt-p53 tumours a high immunoreactivity was revealed with both of the RT-doses, resulting in a clear dose-dependent response. To better characterize the apoptotic pathway, we then performed immunohistochemical staining to assess caspase-3 cleavage, the main executioner of apoptosis (Figure 5, middle column). Quantification of caspase-3 positive cells was performed for all irradiation settings as shown in Figure 5a. Similar to the Bax expression, IR wt-p53 tumours showed a statistically significant difference in the number of caspase-3 positive cells when compared to the CT group (p ⬍ 0.0001). The 20 Gy dose induced a higher apoptotic response when compared to the 10 Gy irradiation group (p ⫽ 0.0331). Of note, a clear dose-dependence for apoptosis was also found in the NIR wt-p53 tumours (20 Gy NIR vs.10 Gy NIR, p ⬍ 0.0001). Apoptotic values in these tumours were also statistically significant when compared to the CT group (p ⬍ 0.0001). Short-term molecular analyses relative to p53-null tumours are shown in Figure 6. Although IR p53-null tumours showed a strong Bax expression, no staining was
observed in NIR p53-null tumours, independent of the dose delivered, similar to CT tumours. After the quantification of caspase-3 positive cells (Figure 6a), IR p53-null tumours showed a significant apoptotic response albeit attenuated with respect to IR wt-p53 tumours. In spite of the lower caspase-3 activation, apoptotic values were statistically significant when compared to their CT for both delivered doses (p ⬍ 0.0001). In contrast, no significant apoptotic response was observed in NIR p53-null tumours after a delivered dose of 10 or 20 Gy. Of interest, values of caspase-3 positive cells were always found to be statistically significant when IR vs. NIR were compared in wt-p53 as well as p53-null tumours. Conversely, similar analysis performed to explore the modulation of the anti-apoptotic bcl-2 molecules in the IR and NIR tumours showed no significant effects in our experimental model (data not shown). The overall results indicate that, although both p53independent and -dependent apoptotic pathways are involved in IR tumour-growth inhibition, the markedly higher caspase-3 activation shown by NIR wt-p53 tumours suggests that the radiation-induced abscopal effect in vivo is strongly dependent on the functional p53 status. In line with this, immunohistochemical staining performed to detect p53 expression showed that p53 positivity increased with the dose in cancer tissues, but was lacking in wt-p53 CT as well as
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Figure 6. Molecular analyses in p53-null tumours 4 h post-irradiation. Representative images of immunostaining with Bax (left column), cleaved caspase-3 (middle column) and p53 (right column). (a) Quantification of cleaved caspase-3 positive cells in p53-null tumours. Representative results of two independent experiments are reported.
the p53-null tumour groups (Figures 5 and 6, right columns). However of relevance, in the NIR wt-p53 tumours the p53 expression was found to be significantly higher upon 20 Gy irradiation while with 10 Gy it was only slightly higher.
Discussion According to the IARC database, 50–70% of human cancers express the wt-p53 gene, depending on the tumour histotype. A plethora of data now available concerning the bystander effect fall into two separate experimental categories, and there is some doubt whether the two groups of experiments are addressing the same phenomenon. First, in vitro experiments involving the transfer of conditioned medium from irradiated wt-p53 and p53-null HCT116 cells showed biologic effects only in non-irradiated wt-p53 cells, suggesting that bystander signals are produced by both wt-p53 and p53-null cells, however only wt-p53 cells respond to them (Mothersill et al. 2011). Secondly, the use of single particle micro beams allows irradiation of specific cells, and thus the study of the biological effects in neighbouring cells. At present, however, only a few number of studies dedicated to explore the abscopal/bystander effect in vivo have been reported. Studies of tumour induction have shown the oncogenic properties of
bystander effects in Patched heterozygous mouse cerebellum (Mancuso et al. 2008). Other studies, have reported the involvement of wt-p53 in the radiation induced bystander antitumour effects, showing NIR tumour-growth delay in wtp53 but not p53-null tumour-bearing mice after irradiation of normal tissue (i.e., right hind leg) (Camphausen et al. 2003). Recently, other studies have shown that combined RT and CTLA-4 neutrilizing antibody promotes radiation induced bystander antitumour effects in NIR tumours (Dewan et al. 2009). Currently, data aimed at assessing the possible role of wt-p53 in radiation-induced bystander effects are contradictory and need to be investigated further. In the present study, through an in vivo experimental approach co-implanting either wt-p53 or p53-null HCT116 cells in both flanks of nude mice and verifying the tumour-growth delay effects upon RT, we were able to observe bystander effects in NIR wt-p53 tumours with a single high dose of 20 Gy delivered to the contra-lateral lesion, demonstrating the relevance of delivered total dose to IR tumours. The results were confirmed also with a different wt-p53 carrying tumour line (data not shown). At the molecular level, short-term analyses, performed on tumours at 4 h postirradiation, showed markedly higher caspase-3 activation in wt-p53 NIR tumours, suggesting that the in vivo radia-
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Abscopal effect in a pre-clinical model of radiotherapy 255 tion-induced bystander effect is principally dependent on the functional p53 status. Conversely, both p53-independent and -dependent apoptotic pathways are involved in IR tumour-growth inhibition. Notably, this study is the first at investigating the effects upon single dose delivery with IORT, where 10 and 20 Gy can be considered equivalent to 20 and 50 Gy delivered at 2 Gy/fraction (conventional fractionation), respectively, assuming an α/β ratio of 10 Gy. Conclusively, our results suggest that the interplay between radiation dose and p53 status plays a critical role in the RT-induced bystander effects (Figure 5). To better assess whether functional p53 protein is required for production of RT-induced abscopal signals in IR cells or bystander effects in NIR cells, similar tumor growth delay studies were performed by co-implanting wt-p53 and null-p53 cancer cells in each flank-side of nude mice and delivering single RT dose (20 Gy) to either wt-p53 or null-p53 lesion. Results, in agreement with in vitro studies (Mothersill et al. 2011), have shown that both IR null- and wt-p53 tumors produce abscopal signals, but functional p53 protein is required to produce bystander effects in NIR lesion (data not shown). Our results are intriguing and could have a wide impact on tailored radiotherapy based on p53 genetic alteration by helping to sterilize nonirradiated tumour cells through bystander signals. In fact, the abscopal effect could be advantageous where the failure after high total doses, such as those delivered in current therapy, in high-risk, i.e., breast or prostate cancer, patients is mainly due to distant disease progression and not to local recurrence.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study was supported by Associazione per la Ricerca sul Cancro (AIRC) with grants to GB (IG#8804) and MM (IG#10357), and TOP IMPLART project (I11J10000420002).
References Bossi G, Marampon F, Maor-Aloni R, Zani B, Rotter V, Oren M, Strano S, Blandino G, Sacchi A . 2008. Conditional RNA interference in vivo to study mutant p53 oncogenic gain of function on tumour malignancy. Cell Cycle 7:1870–1879. Camphausen K, Moses MA , Ménard C, Sproull M, Beecken WD, Folkman J, O’Reilly MS. 2003. Radiation abscopal antitumour effect is mediated through p53. Cancer Res 63:1990–1993. Cividalli A, Creton G, Ceciarelli F, Strigari L, Tirindelli Danesi D, Benassi M. 2005. Influence of time interval between surgery and
Supplementary material available online Supplementary calibration data
radiotherapy on tumour regrowth. J Experim Clin Cancer Res 24: 109–116. Creton G, Benassi M, Di Staso M, Ingrosso G, Giubilei C, Strigari L. 2006. The time factor in oncology: Consequences on tumour volume and therapeutic planning. J Experim Clin Cancer Res 25:557–573. Demaria S, Ng B, Devitt ML, Babb JS, Kawashima N, Liebes L, Formenti SC. 2004. Ionizing radiation inhibition of distant untreated tumours (abscopal effect) is immune mediated. Int J Radiat Oncol Biol Phys 58:862–870. Dewan MZ, Galloway AE, Kawashima N, Dewyngaert JK , Babb JS, Formenti SC, Demaria S. 2009. Fractionated but not single-dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody. Clin Cancer Res 15: 5379–5388. Hainaut P, Hollstein M. 2000. p53 and human cancer: The first ten thousand mutations. Adv in Cancer Res 77:81–137. Jaradat AK , Biggs PJ. 2008. Measurement of the neutron leakage from a dedicated intraoperative radiation therapy electron linear accelerator and a conventional linear accelerator for 9, 12, 15(16), and 18(20) MeV electron energies. Medical Phys 35:1711–1717. Kaminski JM, Shinohara E, Summers JB, Niermann KJ, Morimoto A , Brousal J. 2005. The controversial abscopal effect. Cancer Treat Rev 31:159–172. Mancuso M, Pasquali E, Leonardi S, Tanori M, Rebessi S, Di Majo V, Pazzaglia S, Toni MP, Pimpinella M, Covelli V, Saran A . 2008. Oncogenic bystander radiation effects in Patched heterozygous mouse cerebellum. Proc Nat Acad Sci USA 105:12445–12450. Mole RH. 1953. Whole body irradiation: Radiobiology or medicine? Br J Radiobiol 26:234–241. Mothersill C, Bristow RG, Harding SM, Smith RW, Mersov A , Seymour CB. 2011. A role for p53 in the response of bystander cells to receipt of medium borne signals from irradiated cells. Int J Radiat Biol 87:1120–1125. Ohba K, Omagari K, Nakamura T, Ikuno N, Saeki S, Matsuo I, Kinoshita H, Masuda J, Hazama H, Sakamoto I, Kohno S. 1998. Abscopal regression of hepatocellular carcinoma after radiotherapy for bone metastasis. Gut 43:575–577. Petitjean A , Mathe E, Kato S, Ishioka C, Tavtigian SV, Hainaut P, Olivier M. 2007. Impact of mutant p53 functional properties on TP53 mutation patterns and tumour phenotype: Lessons from recent developments in the IARC TP53 database. Human Mutat 28: 622–629. Shen L, Sun X, Fu Z, Yang G, Li J, Yao L. 2012. The fundamental role of the p53 pathway in tumour metabolism and its implication in tumour therapy. Clin Cancer Res 18:1561–1567. Soriani A , Felici G, Fantini M, Paolucci M, Borla O, Evangelisti G, Benassi M, Strigari L. 2010. Radiation protection measurements around a 12 MeV mobile dedicated IORT accelerator. Med Phys 37:995–1003. Soriani A , Landoni V, Marzi S, Iaccarino G, Saracino B, Arcangeli G, Benassi M. 2007. Setup verification and in vivo dosimetry during intraoperative radiation therapy (IORT) for prostate cancer. Med Phys 34:3205–3210. Strigari L, D’Andrea M, Abate A , Benassi M. 2008. A heterogeneous dose distribution in simultaneous integrated boost: The role of the clonogenic cell density on the tumour control probability. Phys Med Biol 53:5257–5273. Strigari L, Soriani A , Landoni V, Teodoli S, Bruzzaniti V, Benassi M. 2004. Radiation exposure of personnel during intraoperative radiotherapy (IORT): Radiation protection aspects. J Experim Clin Cancer Res 23:489–494.
