Feasibility and guidelines for the use of an injectable fiducial marker (BioXmark ®) to improve target delineation in preclinical radiotherapy studies using mouse models.

Background: Preclinical models of radiotherapy (RT) response are vital for the continued success and evolution of RT in the treatment of cancer. The irradiation of tissues in mouse models necessitates high levels of precision and accuracy to recapitulate clinical exposures and limit adverse effects on animal welfare. This requirement has been met by technological advances in preclinical RT platforms established over the past decade. Small animal RT systems use onboard computed tomography (CT) imaging to delineate target volumes and have significantly refined radiobiology experiments with major 3Rs impacts. However, the CT imaging is limited by the differential attenuation of tissues resulting in poor contrast in soft tissues. Clinically, radio-opaque fiducial markers (FMs) are used to establish anatomical reference points during treatment planning to ensure accuracy beam targeting, this approach is yet to translate back preclinical models. Methods: We report on the use of a novel liquid FM BioXmark ® developed by Nanovi A/S (Kongens Lyngby, Denmark) that can be used to improve the visualisation of soft tissue targets during beam targeting and minimise dose to surrounding organs at risk. We present descriptive protocols and methods for the use of BioXmark ® in experimental male and female C57BL/6J mouse models. Results: These guidelines outline the optimum needle size for uptake (18-gauge) and injection (25- or 26-gauge) of BioXmark ® for use in mouse models along with recommended injection volumes (10-20 µl) for visualisation on preclinical cone beam CT (CBCT) scans. Injection techniques include subcutaneous, intraperitoneal, intra-tumoral and prostate injections. Conclusions: The use of BioXmark ® can help to standardise targeting methods, improve alignment in preclinical image-guided RT and significantly improve the welfare of experimental animals with the reduction of normal tissue exposure to RT.


Introduction
Radiotherapy (RT) remains a critical component of multidisciplinary cancer care.Progress in RT has been driven by advances in RT and imaging technologies with parallel increases in our understanding of RT response at the cell, tissue and whole organism levels.RT relies on image guidance to achieve high levels of precision and accuracy during treatment, most often using computed tomography (CT) to maximise the competing probabilities of tumour control (TCP) and normal tissue complication probability (NTCP).
2][3] FMs can enhance the differentiation of tumour and normal tissue margins in low contrast tissues 4 and their use has significantly improved the accuracy of different RT techniques: intensity modulated RT (IMRT), image-guided RT, volumetric modulated arc therapy (VMAT) and hypo-fractionated stereotactic treatments. 5radiation of tumours and tissues in mouse models requires high levels of precision and accuracy to recapitulate clinical scenarios and limit adverse effects on animal welfare.0][11] These platforms have advanced previous wide field and untargeted RT set-ups, yet the localisation of small, low contrast tissue targets continues to be challenging. 12This has prevented the development of sophisticated and cutting-edge RT protocols, such as hypofractionation (delivering a higher dose of radiation per session in comparison to conventional RT therefore reducing the overall number of treatment sessions), due to limitations in visualisation of some soft tissue targets. 13To overcome targeting errors, lack of standardisation in treatment delivery and improve animal welfare in preclinical models of RT without the need for expensive updated platforms we propose the reverse translation of FMs. 12 FMs are typically composed of high-Z number materials to ensure differentiation from internal structures. 146][17][18][19] In addition, the surgical procedure to insert solid FMs puts mice under high levels of stress and decreases animal welfare. 5,202][23] BioXmark ® has the scope to increase precision and accuracy for the development of innovative preclinical RT studies.Increasing the precision of RT doses can reduce error in treatment targeting and thus reduce the need for large study numbers.5][26] BioXmark ® has been successfully evaluated and deemed advantageous compared to other solid FMs for use in RT treatment in both clinical 22,[27][28][29][30][31][32][33] and preclinical settings. 34,35ccessful refinement of radiotherapy dosing has the potential to impact over 500 mice required annually for radiotherapy studies within the Patrick G. Johnston Cancer Centre, upwards of 4,000 mice per year would benefit nationally, and over 30,000 mice per year internationally.
The purpose of this study is to demonstrate the versatility and ease of use of BioXmark ® in preclinical applications.This article provides guidelines for the injection of BioXmark ® in mouse models to aid visualisation of treatment targets and standardisation of treatment alignment for future preclinical RT treatment set-ups.

Methods
BioXmark ® BioXmark ® is a liquid FM produced by Nanovi A/S (Kongens Lyngby, Denmark).It is a sterile, ready-to-inject liquid composed of biodegradable sucrose acetate isobutyrate (SAIB), iodinated SAIB and ethanol.This formulation ensures that when BioXmark ® is injected into soft tissue the ethanol partly diffuses out of the marker, increasing its viscosity, resulting in the formation of a semi-solid gel. 36BioXmark ® is visible on magnetic resonance imaging (MRI) and ultrasonography due to the SAIB component and visible on X-ray imaging modalities due to the electron-dense iodinated SAIB component. 32Evidence of the visibility of BioXmark ® on multiple imaging modalities in clinical, preclinical and phantom studies is presented in Table 1.
Like other FMs, it is recommended clinically to avoid the injection of BioXmark ® into necrotic tissue, highly vascularised tumour tissue or air-filled cavities. 36Preclinical injection of BioXmark ® should also avoid these tissues and always be through the least invasive procedure possible and actively avoid the need for surgical implantation.Volumes over 50 μl are not recommended for use in mouse models due to negative imaging artefacts on preclinical CBCT scans.An optimum marker volume is suggested between 10-20 μl, this will allow visualisation on CBCT scans without hindering visualisation of small anatomical structures. 35Larger volumes may be more applicable for clinical use or larger animal species.

Animal models
All experimental procedures were carried out in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, and approved by the Queen's University Belfast Animal Welfare and Ethical Review Body (PPL2813).Animals were euthanized by Schedule 1 procedures.Animal studies are reported in compliance with the ARRIVE guidelines. 37l mice were obtained from Charles River Laboratories (Oxford, UK).A mix of mice ages and genders were used due to animal availability from other ongoing studies.Female 12-15-week-old C57BL/6J mice were used for subcutaneous (n=3) and intra-peritoneal injections (n=2), male 14-18-week-old C57BL/6J mice were used for intra-tumoral study (n=21 BioXmark ® injected tumours, n=21 control tumours) and male 10-15-week-old C57BL/6J mice were used for prostate injections (n=2).These numbers were used to assess the feasibility of injection types.No criteria were set to exclude animals from these studies.Mice were randomized to receive either a subcutaneous, or intra-peritoneal injections using an online random sequence generator.Blinding was not possible for intra-tumoral and prostate injections.Blinding was also not possible for image analysis as the injected FM, BioXmark ® , was clearly visualized on CBCT scans.
All mice were housed under controlled conditions (12-hour light-dark cycle, 21°C) in standard caging and received a standard laboratory diet and water ad libitum.To improve the welfare of mice environmental enrichment tools were placed in all cages including cardboard tubes for exploration, softwood blocks to encourage gnawing to prevent teeth overgrowth, nesting material for comfort and mouse swings for added cage complexity and exercise.Mice were also handled gently using refined cupping methods and frequently from a young age to reduce stress.
Mice were anaesthetised with injectable ketamine and xylazine (100 mg/kg and 10 mg/kg) prior to BioXmark ® injection and CBCT imaging.All mice were placed in a heat-box (37°C) for recovery and monitored closely after injections until conscious and returned to normal behaviour.
BioXmark ® uptake A 1 ml micro-dose syringe (Vlow Medical, Netherlands) was used for the uptake and injection of BioXmark ® .Microdose syringes are recommended as they ensure a controlled injection volume.A loading 18-gauge needle was used to fill the syringe slowly from the glass ampoule.BioXmark ® is a viscous liquid so thinner needles may require a longer time to fill the syringe.Air bubbles were checked for through visual inspection prior to injection and were removed by gently tapping the side of the syringe and any excess or leakage BioXmark ® was cleaned using ethanol wipes.The loading needle was removed after uptake into the syringe and replaced with a smaller needle (25-gauge) for each injection.Needles were replaced prior to each injection.
Subcutaneous injection of BioXmark ® BioXmark ® was loaded into a micro-dose syringe as detailed above.For this study, we used a micro-dose syringe with options for the injection of 10, 20 and 40 μl.Mice were anaesthetised with injectable ketamine and xylazine (100 mg/kg and 10 mg/kg) before injection.A small area of fur on each flank was shaved and cleaned with an ethanol wipe for injection.25-gauge needles were used for subcutaneous injections.The skin of the mouse was tented using the thumb and finger and a preselected volume of BioXmark ® (10 μl (n=1), 20 μl (n=1) or 40 μl (n=1)) was injected under consistent pressure.Each mouse received two injections of BioXmark ® , one on each flank, e.g. 10 μl on the left and 10 μl on the right.As BioXmark ® is very viscous the needle was held for 30 seconds after the volume was injected and then slowly removed.All mice were placed in a heat-box for recovery (37°C) and monitored closely until fully recovered from the anaesthetic and then returned to their original cage.
Intraperitoneal injection of BioXmark ® For this study, we used a micro-dose syringe (20 and 40 μl) and 25-gauge needles for injections (20 μl, n=1, 40 μl, n=1).Mice were anaesthetised with injectable ketamine and xylazine (100 mg/kg and 10 mg/kg) before the study, and the injection point sterilized with ethanol before intraperitoneal injection.As BioXmark ® is very viscous, the needle was held for 30 seconds following injection and then slowly removed.No mice presented with bleeding or leakage of BioXmark ® , if there was leakage of BioXmark ® this could be removed with an ethanol wipe.Mice were placed in a heat-box for recovery (37°C) and monitored closely until fully recovered from the anaesthetic before returning to their original cage.

Intra-tumoral injection of BioXmark ®
A previous study reported that it was not possible to mix BioXmark ® with tumour cells and a solid tumour must be established prior to injection of BioXmark ® . 34In this study, we trialled an intra-tumoral injection of BioXmark ® as outlined in Brown et al. 35 Tumour xenograft studies were performed using MC38 colon cancer cells (originate from James W. Hodge) cultured in DMEM media supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin.Cells were maintained at 37°C in a humidified atmosphere of 5% CO 2 and subcultured every 3-4 days to maintain exponential growth.MC38 cells were cultured in vitro and prepared in PBS (1 Â 10 5 cells per 100 μl).Subsequently, 100 μl was injected subcutaneously into the flank of each C57BL/6J mouse (n=42).Mice were anesthetized using inhalant isoflurane (0.5 -2%) for implant and placed in a heat box for recovery.Mice were then returned to conventional housing and closely monitored.Tumour volume was determined three times a week using calliper measurements in three orthogonal dimensions.Once tumours grew to a volume of 100 mm 3 mice were randomised using an online random sequence generator into control (n=21) and BioXmark ® (n=21) cohorts.This injection method was used to assess the radiobiological effect of BioXmark ® with tumours treated with single (16 Gy) or fractionated (2Â8 Gy or 3Â4 Gy) doses of RT.These mice were treated with RT as outlined in Brown et al. 35 For the intra-tumoral injection of BioXmark ® , the FM was loaded into a micro-syringe as detailed above and 25-gauge needles were used for injection.Mice were anaesthetised with injectable ketamine and xylazine (100 mg/kg and 10 mg/kg) and the injection point sterilized with ethanol before injection.The needle was carefully placed into the middle of the tumour (estimated through needle insertion) and 20 μl of BioXmark ® injected under consistent pressure.To prevent leakage or bleeding the needle was held for 30 seconds before removal.No mice presented with bleeding or leakage of BioXmark ® .All mice were placed in a heat-box for recovery (37°C) and monitored closely until fully recovered from the anaesthetic.
Orthotopic prostate injection of BioXmark ® Intra-prostate injections of BioXmark ® were performed under aseptic conditions (n=2).Mice were anaesthetised using inhalant isoflurane (0.5-2%) throughout the procedure and administered analgesia (buprenorphine 0.015 mg/ml (0.05 mg/kg dose)) via IP injection before the surgical procedure and 6 hours post.Protocol adapted from Pavese et al. 38 A small portion skin was shaved and the prostate exposed through a low midline abdominal incision of 5 mm (roughly 1 cm above external genitals).The anterior lobe was identified, and a 26-gauge needle was used to inject 10 μl of BioXmark ® under consistent pressure and the needle held in place for 30 seconds to prevent leakage.Due to the delicate procedure, we trialled a finer gauge needle and found it feasible to inject BioXmark ® effectively with a 26-gauge needle.Successful injection was confirmed by the formation of a small rounding or mound shape.The injection area was checked for bleeding or leakage of BioXmark ® , none was found to leak, before carefully placing the prostate back into the abdominal cavity and the small wound was sealed with tissue glue and stitches.Mice were placed in a heat box for recovery (37°C) and healing, body weight and behaviour were closely monitored after the surgical procedure and then returned to original caging.

Injection protocols BioXmark ® uptake
A. BioXmark ® should be stored at room temperature in a sealed glass ampoule.
C. Carefully open the BioXmark ® ampoule and fill the syringe with the transparent liquid using the loading needle.
-BioXmark ® is a viscous liquid so thinner needles may require a longer time to fill the syringe.
-Remove bubbles by gently tapping the syringe.
-Clean excess or spilled BioXmark ® with an ethanol wipe.
-BioXmark ® is single use, dispose of excess when finished.
D. Once all the liquid has been taken up into the syringe change the loading needle to a new needle for injection (25-gauge or 26-gauge).
E. Fill the new needle with BioXmark ® liquid ensuring no air bubbles are present.
F. Clean any excess BioXmark ® from the needle with an ethanol wipe before injection.

Injection of BioXmark ®
A. Place anaesthetised mouse on a clean surface and shave a small area of fur (if required) on the flank of mouse at the point of implant.
-Once ready for implant suitably restrain, depending on injection route, the mouse for injection.
B. Use an ethanol wipe to clean the skin.
C. Insert the 25-gauge/26-gauge needle (bevel side down) and inject the volume of BioXmark ® required under consistent pressure.Hold for 30 seconds before slowly removing the needle.
D. Check area for bleeding or leakage of BioXmark ® and carefully clean with ethanol wipe if needed.
-If bleeding occurs, check the source of bleeding to ensure no internal organs have been punctured or pain caused to the animal.Apply pressure with clean gauze.If bleeding does not stop take appropriate measure e.g.additional monitoring, pain relief, remove from experiment.
E. If multiple injections of BioXmark ® are required, replace needle and repeat injection process.
F. Place mouse in a heat-box for recovery (37°C) and monitor closely until fully recovered from anaesthetic.

Intra-tumoral injection of BioXmark ®
A previous study trialled mixing BioXmark ® with tumour cells prior to implant for targeting, this was not feasible with the tumour needing to be establish prior to BioXmark ® injection. 34In this study we trialled an intra-tumoral injection of BioXmark ® as outlined in Brown et al 2020. 35Mice were randomised into control of BioXmark ® injected cohorts once sucutaneous tumours reached a suitable size for treatment (100 mm 3 ).This protocol was used to determine the radiobiological effect of BioXmark ® on tumour response, cytotoxicity and dose perturbation. 35For tumour targeting multiple subcutaneous injections of BioXmark ® around the tumour would be more suitable (Figure 1).
A. Ensure needle and syringe are loaded with BioXmark ® and are prepared for injection.-If bleeding occurs, check source and apply pressure to stop.Take appropriate measures if needed e.g.additional monitoring.
F. Place mouse in a heat-box for recovery (37°C) and monitor closely until fully recovered from anaesthetic.

Orthotopic prostate injection of BioXmark ®
Mice were anaesthetised using inhalant isoflurane throughout the procedure and administered analgesia (buprenorphine 0.015 mg/ml (0.05 mg/kg dose)) prior to surgical procedure and 6 hours post.Protocol adapted from Pavese et al 2013. 39 Procedure to be performed under aseptic conditions.
B. Ensure needle and syringe are loaded with BioXmark ® and are prepared for injection.
C. Expose the prostate through a low midline abdominal incision of 5 mm (roughly 1 cm above external genitals).
D. Once the prostate is exposed identify the lobes.
E. Using a 26-gauge needle, inject 10 μl of BioXmark ® into the anterior lobe of the prostate under consistent pressure.
F. To ensure successful injection, check a small bubble had formed.Hold the needle in place for 30 seconds before removal to prevent leakage.
G. Check area for bleeding or leakage of BioXmark ® and carefully clean with cotton tip if needed.
-If bleeding occurs, check source of bleed to ensure no internal organs have been punctured or pain caused to the animal.Use sterile cotton tip to clean the area/apply pressure.Take appropriate measures if needed e.g.additional monitoring, pain relief, remove from experiment.
H. Carefully place the prostate back into the abdominal cavity.
I. Use tissue glue and stiches to close the small wound.
J. Place mouse in a heat box for recovery (37°C).
-Or suitable equivalent e.g.cage placed on heat mat K. Closely monitor mouse after surgical procedure.
-In particular wound healing, body weight and behaviour.

Preclinical imaging
Imaging was completed using the Small Animal Radiation Research Platform (SARRP) (Xstrahl Life Sciences, Camberley UK) onboard CBCT imaging.An imaging energy of 60 kV was used with 0.5 mm Al filtration for all in vivo models.The acquired CBCT scans were transformed into material properties by defining five discrete windows for these materials; air, lung, fat, tissue and bone. 39Imaging was completed immediately after the injection of BioXmark ® , while the mice were still anaesthetised, for subcutaneous, IP and intra-tumoral injections.Mice injected with BioXmark ® into the prostate were allowed to recover overnight in the home cage and imaged the following day.These mice were anaesthetised with injectable ketamine and xylazine (100 mg/kg and 10 mg/kg) for imaging.

CERR analysis
Computational Environment for Radiological Research (CERR) software (https://cerr.github.io/CERR/) was used to complete additional image analysis.Image analysis could not be blinded as BioXmark ® was visible on the CBCT scans.
For contouring of BioXmark ® , all relevant DICOM images and structure sets were exported into CERR software within MATLAB (Version 2019b). 40Structures of interest were contoured manually using the CERR in-house contouring interface.Contours were created slice-by-slice in the coronal plane with corrections made using the sagittal and axial planes.To reduce variations in contours all structures were contoured by the same user.

Statistical analysis
Statistical differences were calculated using unpaired two-tailed student t-tests, or one-way ANOVA tests where appropriate, with a significance threshold of p < 0.05 using Prism GraphPad Prism 7 (Version 7.01, GraphPad Software, Inc.).Data is presented either as the average for the entire experimental arm AE standard error (SEM).

In vivo injection techniques
This study has provided methods for the application of BioXmark ® in small animal models.Mice of both male and female sexes were used and no behavioural or toxic side-effects due to BioXmark ® were observed.
Figure 1A shows CBCT scans from mice injected subcutaneously with BioXmark ® at two separate points on the flank (10, 20 or 40 μl).BioXmark ® is easily visualised in bright white on all CBCT scans and could be easily differentiated from anatomical structures (Figure 1A).Capsules of BioXmark ® formed in the cavity under the skin with all volumes having a similar 3-D shape (Figure 1B & Supplementary Figure 1).Further analysis of the long-term (5-month) stability of BioXmark ® injected subcutaneously is detailed in Brown et al. 35 Due to differences in marker shape between subcutaneous (Figure 1A) and intraperitoneal injections (Figure 2A) we assume that BioXmark ® moulds around anatomical structures before solidifying.There is greater diversity in the shape after injection into the intraperitoneal cavity with the most evidenced by the larger volume of 40 μl (Figure 2A & B), indicating that the intraperitoneal injection is less stable than other methods with the marker breaking up and potentially solidifying in a different area than injected.
Tumour volumes have different shapes and sizes and are heterogeneous causing differences in diffusion of a substance, such as drugs or contrast agents, and potentially BioXmark ® .Figure 3A shows the injection of 20 μl of BioXmark ® into a subcutaneous MC38 colon carcinoma tumour.This was completed to trial the feasibility of injecting BioXmark ® into dense tissue.Intra-tumoral injections were visualised as a hyperdense structure in the centre of the tumour easily differentiated from the tumour tissue (Figure 3A).The injection of 10 μl of BioXmark ® into an orthotopic prostate model is shown in Figure 4A.This method is the most technical and clinically accurate for placement of a FM for RT treatment targeting.The orientation of the marker can be monitored through 3-D analysis providing additional information on the location of the prostate.BioXmark ® can be clearly visualised from CBCT scans but also differentiated from soft tissue through CBCT values for tissue (p=0.0093),air (p=0.0072) and bone (p=0.0073)(Figure 4C).Injected BioXmark ® can help to identify the prostate to improve defining a target volume for beam delivery.This model could be used for normal tissue targeting of the prostate or for targeting orthotopic prostate tumours.

Discussion
This study is the first to provide a detailed methodology for the use of the liquid FM BioXmark ® in preclinical mouse models.The feasibility of multiple injection methods has been trialled for use of the marker as a reference point for submillimetre targeting and beam positioning in preclinical RT set-ups.In this study, we assessed the visibility and 3-D profile of BioXmark ® for subcutaneous, intraperitoneal, intra-tumoral and orthotopic prostate injections.All injection types were feasible and easily performed with BioXmark ® easily differentiated from anatomical structures on CBCT scans.However, the IP injection of BioXmark ® would not be recommended for use in RT targeting due to movement through the IP cavity, it may be more applicable to use a contrast agent for IP injections.We would recommend injection volumes between 10-20 μl of BioXmark ® for use in mouse models.This study complements previous experimental studies for the transferability of BioXmark ® between laboratories to improve treatment targeting in preclinical models of RT. 34,41 When BioXmark ® is injected into soft tissue it forms a semi-solid gel, this acts as an anchor for the FM.Stability is an essential property of FMs for treatment targeting. 14Movement or loss of a marker can lead to inaccurate dose deposition and reduces the efficiency of RT treatment which may be increasingly important during hypo-fractionated schedules. 42Other liquid FMs are not suitable for fractionated RT as they have been reported to not solidify after injection (Lipiodol) or are not stable for long periods (hydrogel). 16,43The iodine-containing contrast agent Imeron 300 has been used preclinically for contrast-enhanced CBCT scans.Dobiasch et al have compared Imeron 300 with BioXmark ® for targeting pancreatic tumours in mice; whilst both were easily visualised on CBCT scans BioXmark ® was concluded more suitable for targeted RT. 34 Imeron 300 has to be continually administered for scanning, adding additional stress on experimental mice. 44BioXmark ® biodegrades slowly with stability shown up to 6 months preclinically. 35De Blanck et al estimate that full marker degradation is up to 3 years clinically 31 ; therefore, BioXmark ® is ideal for outlining treatment parameters for fractionated RT and treatment follow-up.
As a liquid FM, BioXmark ® has advantages over solid FMs with the volume more controllable making it adaptable for preclinical use.The volume of the marker can be target specific and as small as 5-10 μl (Table 1). 34,35Once injected these markers conform to complex shapes surrounding soft tissue or tumour borders for 3-D visualisation.The final shape of injected BioXmark ® can provide crucial information on the shape, size and rotation of a tumour which cannot be achieved with solid FMs. 16,23,45Any changes to the shape of BioXmark ® after irradiation have yet to be reported from current clinical and preclinical studies.We were able to monitor the 3-D shape of intratumorally injected BioXmark ® throughout fractionated treatments (Figure 3B) and showed that the 3-D shape was not significantly affected by 4 Gy fractions of RT.Small changes observed were expected due to changes in tumour shape after irradiation.However, we would recommend using peri-tumoral injections i.e. at multiple points surrounding or adjacent to a tumour, of BioXmark ® for future studies to better align with clinical set-ups and remove BioXmark ® from the treatment volume.
FMs or fixed contrast agents are essential to improve the targeting of tumours in the lower abdomen and prostate in small animal RT.Current set-ups have high levels of errors due to large target fields which reduce the clinical relevance of studies and cause high levels of normal tissue toxicity. 44,46,47In addition, there are significant targeting errors in fractionated treatment schedules due to a lack of stable reference points.Verginadis et al used a solid radio-opaque marker for implantation to the jejunum of a mouse to improve targeting.This study reported increased toxicity levels and blockage of the GI tract. 48BioXmark ® is an alternative marker with no reports of toxic side effects preclinically. 34,35We have demonstrated the ability to use BioXmark ® for the targeting of prostate tumours with clear differentiation of BioXmark ® from anatomical structures (Figure 4C).This approach can be adapted to other orthotopic tumour models to improve the reproducibility of RT targeting and significantly reduce the risk of adverse effects in mice.

Conclusion
Despite advances in preclinical research, only one-third of in vivo studies translate to clinical trials. 49This is largely due to preclinical studies not replicating the clinical setting, and a lack of comprehensive reporting of methods, data sharing and transferability of protocols between laboratories. 50This study shows the feasibility of using BioXmark ® in preclinical models of RT, it's use can be adapted within preclinical radiobiology centres worldwide to improve the visualisation of orthotopic tumours and the translational impact of studies.Standardising the coupling of preclinical RT platforms with targeted imaging tools such as BioXmark ® is a huge step toward quality assurance, reducing targeting uncertainties and most importantly improving animal welfare.

Data availability
Figshare.Preclinical CBCT scans.DOI: https://doi.org/10.6084/m9.figshare.22227490.v1 52is project contains the following data: -CBCT datasets to accompany "Feasibility and guidelines for the use of an injectable fiducial marker (BioXmark ® ) to improve target delineation in preclinical radiotherapy studies using mouse models." Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Reporting guidelines
Figshare.ARRIVE checklist.DOI: https://doi.org/10.6084/m9.figshare.22227478.v1-ARRIVE checklist to accompany "Feasibility and guidelines for the use of an injectable fiducial marker (BioXmark ® ) to improve target delineation in preclinical radiotherapy studies using mouse models." Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).
The study highlights the impact that using BioXmark ® could make on the number of mice used in imaging and radiations studies -cutting down required amount as you are more assured of the accuracy and eliminating poorly targeted tumours.Thus, making the method interesting in terms of the NC3Rs framework.
Methods set out how the study used BioXmark ® in all injection methodologies discussed -intratumoral, intraperitoneal, subcutaneous and orthotopic.Detailed instruction on handling and injecting the reagents are given.Numbers of mice used is given -some experimental numbers used are low but as this is a proof of principle, technical study, are adequate.Both males and females were used, and no toxicity was seen.
Results give a good indication of how the FM would look in preclinical models with examples for each methodology -ruling out the use of intraperitoneal injection due to the disruption in stability of BioXmark ® .They have also shown the volumes of the FM using the CERR software and this shows a 3-D visualisation of the FM.Placement of the BioXmark ® in the orthotopic prostate model proved the most technically challenging but would give good accuracy in dosing the prostatehelping to prove clinical relevance of using this FM in preclinical studies.Long term stability of BioXmark ® has been previously discussed in Brown et.al., reference 35.
Discussion clearly describes how this study has shown the benefits of using this liquid FM in preclinical studies as an aide to increasing targeting accuracy.
An interesting area of study not covered in this research would be to look at how the BioXmark ® interferes with the dose given to the to the tissue -if there was a differential in dose calculated by the preclinical imaging and radiation software -this had been touched on a little in the referenced previous study by Brown et.al and relating the study to improvements in animal welfare.However, in places, particularly paragraph 3, it is difficult to read, due to sub-optimal grammar or punctuation.This would be improved by re-reading and restructuring some of the longer sentences.Methods: In general, the detail in the Methods section is excellent.The only issue is in the section on intra-tumoral injection, which states: 'This protocol was used to determine the radiobiological effect of BioXmark® on tumour response, cytotoxicity and dose perturbation (35).Reference 35 (Brown et al 2020), is frequently referred to throughout the text, but relates to a different tumour cell line, Lewis Lung, rather than the MC38 cell line described here.To me, there is insufficient data on how these two studies are related.It would be helpful to add a bit more information and clarification on the two studies, if appropriate, to assist the reader.Preclinical imaging method section: "An imaging energy of 60 kV was used" -keV is not a unit of energy Results: The Results presented are quite straightforward; firstly a characterization of BioXmark injection at subcutaneous (n=3), intra-peritoneal (n=2)and intra-prostate (n=2) sites; with methodological recommendations proposed for each location.The main contents of the Results show the appearance of the marker in whole mouse CT scans and a contour-derived representation of BioXmark distribution from within each tissue (Figures 1, 2 and 4).These sections provide useful information for other users, but did feel a bit limited.I would prefer that there was a summary of the effects of BioXmark on dose calculation at these locations, and if possible some information on the effect on measured radiation dose, or a reference to these, if indicated in an additional publication.It would be helpful to have confirmation that the Muriplan software can calculate accurate doses in regions containing the BioXmark, and that if this is an issue, how best it could be mitigated.However, this notwithstanding, the study provides a lot of detail on injection protocols that will be useful for researchers looking to use Bioxmark as an FM in pre-clinical irradiations.It is also informative as it shows the relative utility of BioXmark for injection at different sites.The remaining Results section, referring to Figure 3, details intra-tumoral injection (n=42) to demonstrate 'the feasibility of injecting BioXmark® into dense tissue.' and shows CT scans from one mouse and a representation of BioXmark distribution over the course of a 4x 4 Gy irradiation schedule, to highlight the minimal changes to BioXmark following irradiation.For this section, the timescale over which the 4x4 Gy fractions were delivered should be added.Also, there is a statement that 'no radiobiological effects of the BioXmark were determined, further detailed in reference 35.'However, reference 35 presents very limited detail on radiobiological effect, only dose calculation discrepancies and the impact that these may have on tumour growth following treatment.Therefore it might be best to restate in this current paper, the potential impact on dose calculation of BioXmark in the target volume.If there are any biological markers of radiation effect that have been analysed, in the presence/absence of BioXmark, following treatment, it would be good to mention that here.If there is n=42 data, these should be presented.

Introduction
The authors include a good introduction of the use of FMs, with the limitations of using solid FMs and how liquid FMs are more suitable in the RT context.Are there any limitations to using liquid FMs? 1.

Methods
Table 1 is a good summary of the current uses of BioXmark but it is quite difficult to distinguish the subheadings within the table as they blend in with the other rows. 1.
Paragraph 2 line 1 -can the authors describe why it is not recommended to inject BioXmark into necrotic tissue, highly vascularised tissue or air cavities and is there a way to avoid this? 2.
In accordance with the ARRIVE guidelines can you include: i) The total number of mice used for the study -how many of these were from other studies?ii) How were the sample sizes calculated?iii)Were there any animals/data points not included in the analysis? 3.
The authors describe the optimal volume to be 10-20ul, was there a particular reason why 10ul wasn't used for the intraperitoneal site and 40ul was used for the subcutaneous and intraperitoneal sites? 4.
Can the authors include the suppliers of all consumables: needles, cell culture reagents etc? 5.
What radiobiological effects were assessed in the intra-tumoral group?6.
For the fractionated group, could you please clarify if these were on consecutive days?7.
"Injection of BioXmark protocol" -Is this for the subcutaneous and intraperitoneal injection sites?

8.
Preclinical imaging -what software was used to define the material properties?Have you used the right reference here?9.

Results
Figure 3B -can you clarify that the different doses in the figure are accumulations of the individual fractions. 1.
I would suggest adding details of the volume contouring to figure captions 2 and 4, to be consistent with figure captions 1 and 3.

2.
Can the last two sentences of the final paragraph be combined as there is slight repetition?Would it sound better as "normal tissue sparing of the prostate" as it currently may sound like you are looking to target the prostate with radiation rather than identify it to make sure it is out of the radiation field? 3.

Discussion
Paragraph 2, sentence 3 -to avoid confusion would it be better to say "movement or loss of a marker can lead to inaccurate targeting" rather than dose deposition as this reads like the FM deposits the dose. 1.

Are the 3Rs implications of the work described accurately? Yes
Are a suitable application and appropriate end-users identified?Yes

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Yes Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Radiobiology, preclinical dosimetry I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
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Table 1 .
Combination of clinical, preclinical and phantom studies published outputs stating the visibility of different volumes of BioXmark ®

B
. Place anaesthetised mouse on a clean surface and use an ethanol wipe to sterilise the point of injection.C. Insert the 25-gauge/26-gauge needle (bevel side down) to the middle of the tumour.D. Inject the desired volume of BioXmark ® (10-25 μl) under consistent pressure and wait 30 seconds before removing the needle.E. Check area for bleeding or leakage of BioXmark ® and carefully clean with ethanol wipe if needed.

Figure 1 .
Figure 1.In vivo CBCT imaging analysis of the different volumes of BioXmark ® following subcutaneous injection.Panel A: CBCT scans of 10 (i), 20 (ii) and 40 µl (iii) of BioXmark ® (left to right) injected subcutaneously at two points on the flank of mice (n =1).Panel B: Volumes of BioXmark ® on CBCT scans were contoured using CERR software and 3-D shape of each marker visualized.
3-D reconstructions of injected BioXmark ® which underwent fractionated RT (3Â4 Gy) are shown in Figure 3B.Minimal changes were observed in the shape of BioXmark ® .No radiobiological effects of BioXmark ® were determined, further detailed in Brown et al.35

Figure 3 .
Figure 3.In vivo CBCT imaging analysis of the intra-tumoral injection of 20 µl of BioXmark ® .20 µl was injected into MC38 subcutaneous tumours once they reached a volume of 100 mm 3 .Panel A: Mice were imaged on CBCT at 60 kV prior to receiving radiotherapy (n=21).Panel B: Volumes of BioXmark ® on CBCT scans were contoured using CERR software and the 3-D shape of each marker was visualised throughout the dose schedule.

Figure 4 .
Figure 4.In vivo CBCT imaging analysis of the prostate orthotopic injection of 10 µl of BioXmark ® .Panel A: Mice were imaged on CBCT at 60 kV after injection of BioXmark ® (n=2).Panel B: 3-D shape of injected BioXmark ® .Panel C: Viability of the marker was quantified through CBCT numbers for BioXmark ® , tissue, air and bone.Data presented are mean values AE SEM; statistical significance is reported as *< 0.05, ** < 0.01.

the 3Rs implications of the work described accurately? Yes Are a suitable application and appropriate end-users identified? Yes Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests:
., reference 35.It would have been good to have details of the scheduling of the fractionated doses of radiation in the methods/results sections.Methods/protocols sections -the text is quite repetitive.No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.The title of this manuscript summarises the content well: Mainly, the paper concerns development of injection protocols for a liquid fiducial marker (BioXmark) that can be used preclinically during CT imaging and radiotherapy.Abstract:.please correct:..... to ensure accuracy of beam targeting.(new sentence) This approach ..... Introduction: The Introduction does a good job of setting the scene for this study, outlining use in relation to clinical applications, describing the problems inherent in using solid FMs preclinically, © 2024 Sosabowski J et al.

the 3Rs implications of the work described accurately? Yes Are a suitable application and appropriate end-users identified? Yes Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes
Discussion and Conclusion: These were very clear and concise Additional issuesThe text is a bit repetitive in places, also one section occurs twice, headed: 'Intra-tumoral injection of BioXmark' , and the first two sentences starting: 'A previous study trialled mixing BioXmark® with tumour cells prior to implant for targeting…' Therefore, one of the sections should be deleted.Statistical analysis is not particularly necessary but is provided to show the difference in CBCT values for radiodensity between the BioXmark and other tissues on the CT scan.In my opinion, it is not necessary to give exact p-values to multiple decimal places for these p-values, as large numbers of decimal places are harder to read.The test used to generate the p-values should be indicated.Please adjust this if possible.
Competing Interests: No competing interests were disclosed.