A High-Throughput In Vitro Radiobiology Platform for Megavoltage Photon Linear Accelerator Studies

Round 1

Reviewer 1 Report

Innovative

Author Response

We would like to thank the reviewer for their time in reviewing our manuscript and feedback.

Reviewer 2 Report

This manuscript describes a method to design and develop a multiwell tissue culture plate irradiation setup，which supports 96，24 and 6 well tissue culture plates for the Intensity Modulated Radiotherapy and open-field, homogenous irradiation. The spatial distributed doses 0,2,4,8,16 and 24 Gy can be easily controlled and realized for different tissues irradiation treatments by the facilities such as Cobalt-60, advanced MV photon source or high intensity linear accelerators. The experimental results for the A375 and LN18 line cells are very interesting. It shows the success of the multicell tissue culture plate, it is really high-throughput in-vitro screening of irradiation-drug combination within a clinical set-up.

    The only regret is the difference between predicted dose of 24Gy and real spatial distributed doses for different tissue culture plate. The maximum and minimum dose are a bit far from the mean value. This may be improved for the more accurate dose delivery irradiation in the next step. Hope the authors could find the way to make it better.

Author Response

We would like to thank the reviewer for their time reviewing our manuscript and feedback. We carefully considered all of their comments and our point by point response provided in red font below.

 

This manuscript describes a method to design and develop a multiwell tissue culture plate irradiation setup，which supports 96，24 and 6 well tissue culture plates for the Intensity Modulated Radiotherapy and open-field, homogenous irradiation. The spatial distributed doses 0,2,4,8,16 and 24 Gy can be easily controlled and realized for different tissues irradiation treatments by the facilities such as Cobalt-60, advanced MV photon source or high intensity linear accelerators. The experimental results for the A375 and LN18 line cells are very interesting. It shows the success of the multicell tissue culture plate, it is really high-throughput in-vitro screening of irradiation-drug combination within a clinical set-up.

Thank you for the comment. We utilized a widely used prototype clinical linear accelerator to demonstrate that in-vitro experiments are possible with existing clinical radiotherapy infrastructure. Agree with the reviewer, with more advance machines with features such as very fine multi-leaf collimators would help further improve our platform in the future.

 

The only regret is the difference between predicted dose of 24Gy and real spatial distributed doses for different tissue culture plate. The maximum and minimum dose are a bit far from the mean value. This could be improved with more accurate dose delivery irradiation in the next step. Hope the authors could find the way to make it better.

We agree with reviewer comment. We aimed to generate the best plan for each plate but given the differences in their geometry, there were differences in spatial dose distribution between plates, which as mentioned in the above response could be improved in future with a better machine.

Reviewer 3 Report

Abstract and Introduction

Remove IMRT from the abstract, wrote full form, readers cannot understand in short form when it come such early in abstract.

How author envision to in vitro in vivo extrapolation of the method developed for the real time cancer radiotherapy? What readout author can think about to help the track radiotherapy in real cancer radiotherapeutic regimes? In this context, putting a concluding statement in the abstract will add value to this work.

In Introduction, Cite a latest report https://doi.org/10.1021/acs.chemrestox.1c00219 to refer the sentence ending with ‘large tissue culture vessels and laboratory irradiators are widely used for this purpose’, which emphasizes how advances in in vitro methods could potentiate prediction model for in vivo therapeutic and toxicological effects.

While enumerating various advantages, I suggest authors also put some important for such in vitro validated HTP platform development is useful because 1) in vitro HTP models allow the use of cells from human sources, avoiding possible inter-species differences related to animal models; 2) Such in vitro radiotherapy model rae useful since the Ionizing radiation (IR) damage to endothelial cells or ECs composing the microvasculature is particularly troublesome since ECs are present both in tumor and healthy tissues, playing an essential role in shaping the microenvironment; 3) Such in vitro platforms are key to obtaining information on potential molecular targets for the setting up of novel radiosensitization approaches or for the design of mathematical algorithms suitable for tailoring radiotherapy as shown lately https://doi.org/10.1007/s11684-020-0761-1.

Materials and Methods

Provide catalogue number and source of all materials and model/version supplier of all instruments used in the study.

For the accurate in vitro analysis using different imaging and Chemianalytics platform in tissue culture plate (6, 24, 96 well), the design accuracy is paramount important for calibration and standardizing the experiment. Therefore, the Plate irradiation setup development needs in depth info. I encourage authors to provide CAD model with full details in materials method, as authors give a cartoon in figure 1.A is not sufficient.  Provide dimension details and design parameters for 3 types of culture plate tested in this report.

Are clinical linear accelerator automated as shown in fig.1 C-D or authors have to adjust the platform manually?

Fig. 1 caption “Plates positioned with aid of multiwall tissue culture plate set-up and room lasers.”, please rephrase as it is unclear.

 Results and discussion

Are wells filled with any liquid as shown In Figure 2. A, B & C. Coronal view of 96, 24 and 6 well tissue culture plates showing contouring of wells? Do authors think the flattening meniscus of well from 96 to 6 well can make any imaging artifacts which could be in decisive for the analysis?

Statement in discussion section ‘In order to screen the large number of potential radiation-drug combinations quickly and efficiently, there is a need for a high-throughput, in-vitro radiobiology platform’ needs a suitable reference, cite a Latest report  doi:10.15761/FNN.1000S2006 regarding HTP platforms changing the landscape of clinical research in this context.

In Table 1. Radiation dose statistics of multiwall tissue culture plate IMRT plans, how the ‘0’ Gy target dose wells have some numbers in mean/max/min calculated, please clarify it is confusing.

Give details how ‘predicted’ doses were calculated, did author used and mathematical prediction tools, provide details please?

Provide high-resolution plots with bigger font size to make data readable. Data visualization is really poor quality.

Add scale bars to all images.

 

 

Comments for author File: Comments.pdf

Author Response

We would like to thank the reviewer for their time reviewing our manuscript and feedback. We carefully considered all of their comments and our point by point response provided in red font below.

 

Remove IMRT from the abstract, wrote full form, readers cannot understand in short form when it come such early in abstract.

This change has been made. Thank you.

 

How author envision to in vitro in vivo extrapolation of the method developed for the real time cancer radiotherapy? What readout author can think about to help the track radiotherapy in real cancer radiotherapeutic regimes? In this context, putting a concluding statement in the abstract will add value to this work.

Thank you. We believe with the option of 3 different tissue culture plates, various ex-vivo experiments can be performed, such as cell-viability, clonogenic assays, PCR. A sentence reflecting this is added to the end of the abstract.

 

In Introduction, Cite a latest report https://doi.org/10.1021/acs.chemrestox.1c00219 to refer the sentence ending with ‘large tissue culture vessels and laboratory irradiators are widely used for this purpose’, which emphasizes how advances in in vitro methods could potentiate prediction model for in vivo therapeutic and toxicological effects.

Referenced, thank you.

 

While enumerating various advantages, I suggest authors also put some important for such in vitro validated HTP platform development is useful because 1) in vitro HTP models allow the use of cells from human sources, avoiding possible inter-species differences related to animal models; 2) Such in vitro radiotherapy model rae useful since the Ionizing radiation (IR) damage to endothelial cells or ECs composing the microvasculature is particularly troublesome since ECs are present both in tumor and healthy tissues, playing an essential role in shaping the microenvironment; 3) Such in vitro platforms are key to obtaining information on potential molecular targets for the setting up of novel radiosensitization approaches or for the design of mathematical algorithms suitable for tailoring radiotherapy as shown lately https://doi.org/10.1007/s11684-020-0761-1.

Thank you for suggesting these additional benefits, which have been added as a new paragraph in the introduction section (page 2, para 4) and suggested paper is referenced.

 

Materials and Methods

 

Provide catalogue number and source of all materials and model/version supplier of all instruments used in the study.

Additional information where available provided in the materials and methods section. Most materials/manufacturer details have been identified with supplier details for laboratory reagents. Due to authors move to other institutions, we are unable to provide some information due to lack of access. We believe and are confident our experiments can be replicated successfully with details provided in the revised manuscript.

 

For the accurate in vitro analysis using different imaging and Chemianalytics platform in tissue culture plate (6, 24, 96 well), the design accuracy is paramount important for calibration and standardizing the experiment. Therefore, the Plate irradiation setup development needs in depth info. I encourage authors to provide CAD model with full details in materials method, as authors give a cartoon in figure 1.A is not sufficient.  Provide dimension details and design parameters for 3 types of culture plate tested in this report.

Thank you.  More details about the tissue culture plate set-up added as requested in page 3, para 1 and in figure 1 foot note. Main feature of tissue culture plates added, and full finer details are easily accessible from main commercial suppliers.

Are clinical linear accelerator automated as shown in fig.1 C-D or authors have to adjust the platform manually?

This is a fully automated linear accelerator and once the plates were set-up within the plate-holding device using room lasers, IMRT plans delivered remotely without any manual adjustment to the platform or to the accelerator.

 

Fig. 1 caption “Plates positioned with aid of multiwall tissue culture plate set-up and room lasers.”, please rephrase as it is unclear.

Rephrased as advised. Thank you.

 

Results and discussion

 

Are wells filled with any liquid as shown In Figure 2. A, B & C. Coronal view of 96, 24 and 6 well tissue culture plates showing contouring of wells? Do authors think the flattening meniscus of well from 96 to 6 well can make any imaging artifacts which could be in decisive for the analysis?

Wells filled with water to simulate the amount of tissue culture medium used when in-vitro experiments using cell lines performed as part of the study. Although there is flattening of the meniscus, this would not affect dose delivery/deposition as an advance radiotherapy technique; IMRT was used to generate plans, which produced satisfactory dose distribution to the columns of water as shown in table 1 and delivery confirmed with dosimetry.

 

Statement in discussion section ‘In order to screen the large number of potential radiation-drug combinations quickly and efficiently, there is a need for a high-throughput, in-vitro radiobiology platform’ needs a suitable reference, cite a Latest report  doi:10.15761/FNN.1000S2006 regarding HTP platforms changing the landscape of clinical research in this context.

Thank you. Reference added.

 

In Table 1. Radiation dose statistics of multiwall tissue culture plate IMRT plans, how the ‘0’ Gy target dose wells have some numbers in mean/max/min calculated, please clarify it is confusing.

This was due to ‘dose spillage’ during IMRT planning and optimization to meet our planning objectives that we set out (page 4, para 1). More advanced, newer accelerators may have much finer multi-leave collimators which would help to minimize/avoid this.  

 

Give details how ‘predicted’ doses were calculated, did author used and mathematical prediction tools, provide details please?

For radiochromic dosimetry, standard guidelines/methodology described in ref 10 was used to calculate predicted doses. For EPID predicted doses, standard methods were used and as per manufacturer’s (Varian Truebeam) specifications used, additional ref, ref 11 added for detailed information.

 

Provide high-resolution plots with bigger font size to make data readable. Data visualization is really poor quality.

Thank you. All figures replaced by better/bigger/hi-res images. Fig 3 split into three to make scale bars more visible as they were screen shots taken from different systems.

 

Add scale bars to all images.

All figures reviewed for appropriate scale bars and amended as needed to improve presentation and readability.

 

 

 

 

 

Round 2

Reviewer 3 Report

accept