A New Low-Energy Proton Irradiation Facility to Unveil the Mechanistic Basis of the Proton-Boron Capture Therapy Approach

Round 1

Reviewer 1 Report

With the present manuscript entitled “A new low-energy proton irradiation facility to unveil the mechanistic basis of the Proton-Boron Capture Therapy approach” the authors implemented a novel setup for radiobiology experiments at a 3-MV tandem accelerator. It is a very interesting manuscript describing the enhancement in cancer cell killing due to the 11 B carrier BSH, thereby corroborating the mechanistic bases of Proton-Boron Capture Therapy. It sheds light to fundamental mechanisms of the biological action of charged particles with the aim to potentiate the therapeutic capabilities of protontherapy.

It is well written and comprehensive.

Some minor comments are provided:

1. The authors should mention whether they had control samples -unirradiated but treated with BSH- in order to take into account a probable toxicity of BSH. (This is not clear in Fig 6 and 7).
2. The authors could also explain the reason why they chose the specific cell lines and briefly discuss their radiosensitivity.
3. p11 lines 354-367: It would be better if this part was moved to the discussionsection.
4. Some few minor comments about abbreviations:

p.2 line 71: The abbreviation SOBP should be explained (Spread out Bragg Peak) as this is the first time it appears in the text.

p.9 line 321 The abbreviation DMF (Dose Modifying Factor) -though it is explained in the tables 2 and 3 below-  should also be explained in the text, as this is the first time it appears in the manuscript.

 

Author Response

The authors would like to thank the reviewer for his/her comments, which are punctually addressed below.

1. We thank the reviewer for pointing out this important aspect, which is now addressed in the revised version of the manuscript in both the Materials and Methods and in the Results Sections. In accord with previous experiments conducted at other facilities (i.e. INFN-LNS, Catania, Italy and CNAO, Pavia, Italy), we found no cytotoxicity induced by BSH alone. Thus, as requested by the reviewer, it is now explicitly stated in the Materials and Methods Section (paragraph 2.5) that unirradiated samples treated with BSH were used to take into account the possible effect by the boron carrier irrespective of radiation; in addition, in the Results Section (paragraph 3.2), we have reported the values found for the Plating Efficiency (PE), which by definition measures the proliferative ability of unirradiated controls and against which the Surviving Fractions reported in Figures 6 and 7 are normalized. Such values were identical for both cell lines between BSH-treated and untreated samples.

 

2. We agree that the rationale for choosing the specific cancer cell lines used in this work is an important issue. However, this work essentially represents the continuation and extension of previous experiments on the PBCT, particularly as regards the DU145 prostate cancer cell line as pointed out throughout the manuscript. These were initially used in PBCT proof-of-principle experiments (Cirrone et al, ref. 16) as protontherapy is being explored for localized prostate cancers. In this manuscript, we wanted to shed light on the mechanistic basis of PBCT by developing a beamline that allowed us to irradiate cells with protons of energies close to those of the cross-section maximum. Indeed, for the first time we also irradiated cells from the PANC-1 prostate cancer cell line, whose radioresistance is well known and referred to in paragraph 3.2 of the Results Section, where we also highlight that the radiosensitizing effect of the BSH due to the proton-boron (pB) reaction is slightly less, yet significant, compared to that measured for the DU145 cells. Such a difference is actually mentioned also in the second half of the Discussion Section, where we imply the possible clinical relevance that the ability of the pB reaction may bear for protontherapy to be able to overcome cancer radioresistance. In fact, we have accumulated data showing that PBCT is able to increase cell death of PANC-1 cells also at high-energy, clinically used proton beams, which we are in the process of submitting.

3. We appreciate the reviewer’s observation. However, we do feel that the part she/he refers to is probably best suited to the Results Section, both to avoid some degree of repetition with what is now present in the Discussion Section as regards the radiobiological part of the work as well as to properly highlight the relevance of the work carried out regarding the beamline design and implementation. Should the reviewer insist, we shall re-consider this point, of course.

4. We have made the acronyms SOBP and DMF explicit in the Introduction (page 2) and in the Results Section (page 9) as rightly requested by the reviewer.

Reviewer 2 Report

I enjoyed reading this manuscript.  Very interesting!  Should be published with a high priority.

Author Response

The authors wish to thank the reviewer for her/his time and the very positive remarks on the manuscript, which we gladly appreciate.

Reviewer 3 Report

In the present work, to realize a new radiation therapy based on proton-boron fusion reaction, a new irradiation system with a 3 MeV proton accelerator has been developed and preliminary experiment has been done. This is a first step to the actual treatment of Proton-Boron Capture Therapy, PBCT. Thus, this report would be published in Applied Sciences. However, the manuscript should be slightly revised according to the comments below.

(1) More detailed description on the physics of proton-boron(¹¹B) fusion reaction such as cross-section of the reaction as a function of proton energy, energies of produced a-particles, accompanied gamma rays, and so on is highly expected.

(2) Describe the distribution of BSH inside the cell and outside the cell, substrate.

(3) p.2 line 96: (SSNTDs) should be (SSTDs) because of the descriptions in the following Figures, Tables and text.

Author Response

We thank the reviewer for his positive comments. As for the points raised, please see punctual response below.

(1) We appreciate the expectations on a more detailed description of the physics underlying the proton-boron (pB) reaction expressed by the reviewer. However, as this work represents the extension of previously published experimental results on the Proton-Boron Capture Therapy (PBCT) approach, to which appropriate references are provided throughout the text, we felt that details such as those mentioned by the reviewer are therefore already readily available. The pB reaction, as the reviewer of course is aware of, has been well-characterized in the nuclear physics context. Indeed, a graph depicting the relationship between cross-section and incident proton energy, reproducing existing nuclear physics database, was published in our first work (see Cirrone et al, ref. 16). Moreover, we explicitly stated that the maximum value for such cross section occurs at the energy at which the biological samples were for the first time irradiated with a monoenergetic proton beam, which constituted the very rationale for the radiobiology part of the work here presented. Similarly, well-known (and punctually cited in the previous experimental reports to which we refer in this work) are the energy distribution spectra of the emitted alpha particles and the prompt-gamma emission. Given the nature of the Applied Physics journal and the requirement of describing the background of our work in a succinct manner able to reach an audience as wide as reasonably possible (hence not necessarily familiar in highly specialised fields such as nuclear physics or radiation biology), we felt that repeating these details could have been avoided.

(2) We thank the reviewer for highlighting this point. PBCT is inspired by the much older Boron-Neutron Capture Therapy (BNCT) in that they share the same nuclear physics reaction-based binary approach and indeed the use of boron carriers (albeit the use of the other isotope in PBCT, i.e., 11B, is envisaged instead of the 10B used in BNCT). In fact, to establish whether the PBCT is clinically feasible, we started with the oldest boron compound used in BNCT, that is BSH. Also in this case, the poor internalization of BSH within living cells is amply documented in the BNCT literature compared to metabolically actively incorporated compounds such as BPA (which we have been testing and whose results we are in the process of submitting). However, to prove that PBCT can potentially augment the biological effectiveness of protontherapy, we thought, as argued in previous work, that BSH would best suited because of its richness in boron (12 atoms), and hence in the abundance of the 11B isotope. Therefore, in these experiments cells were pre-treated with BSH in agreement with BNCT-based protocols, using the most widely adopted nominal concentration (80 ppm of 11B), but cells were always irradiated in the presence of BSH: during irradiation, cells were kept in the BSH-containing medium. it follows that exact distribution of BSH was not measured since the priority was whether a radiosensitizing effect could be observed, irrespective of the localization of BSH. On the other hand, BSH not been incorporated by the cell but lying on its membrane would still emit, if the reaction is triggered, alpha particles capable of hitting the DNA, given the known emission energy (around 4 MeV), and hence, range in tissue (some 20 microns). Indeed, contrary to BNCT, where selective incorporation of boron in cancer cells as opposed to normal cells is crucial-and is one of the major difficulties in establishing BNCT as a routine clinical treatment-in PBCT it would be the physics to drive the process. The reaction in vivo would, in fact, occur only where the tumour lies since it is there that the SOBP, and therefore the slowing-down protons, would be placed, making, in principle, less stringent the requirement for boron internalization.

(3) We amended as rightly pointed out by the reviewer.

Reviewer 4 Report

This paper describes the development of beamline for radiobiology experiments at a 3-MV tandem accelerator to apply the experiment for proton-boron capture therapy (PBCT).  And the authors elucidated the tumor cell killing effect of BSH by PBCT.

These results are very interesting and important for radio biology.

However, the detail of biological experimental data is lacked in this manuscript.  The authors must describe the detail of colony assay. Especially, cell-culture condition of after irradiation such as seeding out cell count, used cell culture plate and cultured time (about each cell line).

In this reviewer opinion, to accept this paper for Applied Sciences, I think this manuscript lacks some experimental data.

 

Author Response

We appreciate the concern raised by reviewer regarding the lack of experimental details on the colony-forming assay, which basically reflects the protocol first proposed in 1956 by Puck and Marcus. In fact, given the scope of the Journal to try and reach a wide audience, we saw it appropriate to be rather concise on this part, obviously accompanying its description with the opportune citations. Several details on the more biological procedures related to cell culture handling and sample preparation for the clonogenic assay were already provided in the first version of this manuscript. We nevertheless have now added some more information in the corresponding part of the Materials and Methods Section, that is paragraph 2.5. We trust this be satisfactory for the reviewer, whom we thank for his/her time in revising our work.