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Published in final edited form as: Phys Med Biol. 2020 Aug 19;65(16):165008. doi: 10.1088/1361-6560/ab9776

Radiodynamic therapy using 15-MV radiation combined with 5-aminolevulinic acid and carbamide peroxide for prostate cancer in vivo

Joseph V Panetta 1, Dusica Cvetkovic 1, Xiaoming Chen 1, Lili Chen 1, C-M Charlie Ma 1
PMCID: PMC8409498  NIHMSID: NIHMS1733676  PMID: 32464613

Abstract

Photodynamic therapy has been clinically proven to be effective, but its effect is limited to relatively shallow tumors because of its use of visible light. Radiodynamic therapy (RDT) has therefore been investigated as a means to treat deep-seated tumors. In this study, the treatment effect of a novel form of RDT consisting of radiation combined with 5-aminolevulinic acid (5-ALA) and carbamide peroxide was investigated using a mouse model. Male nude mice were injected bilaterally and subcutaneously with human prostate cancer (PC-3) cells and randomized into 8 treatment groups, consisting of various combinations of 15-MV radiotherapy (RT), 5-ALA, and carbamide peroxide. The treatment effect of a single fraction of treatment was measured by calculating tumor growth delay, monitored using weekly MR scans. The ability of the drugs to be delivered to the tumors was qualitatively measured using 18 F-FDG PET/CT scans. RDT was shown to significantly delay the tumor growth for the mouse model and tumor cell line investigated in this work. Tumors treated with RDT showed a decrease in tumor growth of 24 ± 9% and 21 ± 8% at one and two weeks post-treatment, respectively. Peroxide and 5-ALA did not contribute significantly to tumor growth delay when administered alone or separately with RT. Blood perfusion was shown to be able to deliver agents to the tumors investigated in this work, although uptake of 18 F-FDG was shown to be non-uniform.

Keywords: radiodynamic therapy (RDT), photosensitizer, radiotherapy, prostate cancer, MRI, PET/CT, tumor growth delay

1. Introduction

Photodynamic therapy (PDT) is a well-studied treatment modality and has been clinically proven to be an effective treatment for several cancer types, including skin, bladder, and esophageal cancer (Zheng 2005, Qumseya et al 2013). The mode of action by which PDT works to eradicate tumors requires the presence of oxygen and involves the absorption of photons by the photosensitizer to activate it to an excited state from its ground state. This energy is ultimately transferred to produce oxygen singlet species (1O2) and reactive oxygen species (ROS), which lead to cellular death predominantly by three mechanisms: direct tumor death (e.g. necrosis, apoptosis), damage to tumor vasculature, and activation of a host immune response (Ketabchi et al 1998, Hashiguchi et al 2002, Davids et al 2008, Takahashi and Misawa 2009, Agostinis et al 2011, Castano et al 2015, Takahashi et al 2016, Hwang et al 2018). Photosensitizers and the drugs that deliver them to tissue have been developed over the last several decades and several are FDA approved, including 5-aminolevulinic acid (5-ALA) (Zheng 2005, Agostinis et al 2011, Yoon et al 2013). The properties of this drug have been well characterized, including the uptake and clearance of 5-ALA in tumor tissue, as well as the absorption spectrum and the photocytotoxicity of PPIX (Kennedy et al 1996, Marcus et al 1996, Peng et al 1997, Ketabchi et al 1998, Moan et al 2002, Lawrence et al 2014).

PDT photosensitizers are most commonly irradiated with visible light, limiting treatment to relatively superficial, localized tumors. The ability of high-energy radiation to activate the photosensitizers in radiodynamic therapy (RDT) is therefore a subject of active investigation, since high-energy x-ray and gamma ray photons can penetrate deeply into tissue (Axelsson et al 2011, Glaser et al 2015, Hartl et al 2015, 2016). The activation of PPIX by Cherenkov radiation produced in tissue by external beam radiation has been shown, and studies have shown that radiation produced by linacs is sufficient to activate enough PPIX to cause cellular damage (Hashiguchi et al 2002, Axelsson et al 2011, Takahashi et al 2013, Glaser et al 2015, Takahashi and Iwahashi 2017, Larue et al 2018). Some studies have focused on RDT using kV or low-MV x-rays combined with 5-ALA; these works have shown a delay in tumor growth using mouse glioma and melanoma models, with the results supported by in vitro studies with similar energies, indicating that 5-ALA may be activated at these energies as well (Henderson et al 2006, Yamamoto et al 2012, 2015, Takahashi et al 2013). While the exact mechanism by which radiodynamic therapy leads to tumor destruction has not been investigated thoroughly, studies have shown that activated photosensitizers may lead to tumor destruction by both necrosis and apoptosis (Larue et al 2018). Moreover, the relative doses of activating radiation and 5-ALA are important to determining the cytotoxic effect, and the optimal time to treat after administration of 5-ALA has been investigated (Zheng 2005, Jarvi et al 2012, Takahashi et al 2013, 2015, 2018, Larue et al 2018). Studies have also investigated the potential of hydrogen peroxide to be an adjuvant to radiotherapy by enhancing cellular radiosensitivity (Fang et al 2013, Rosli et al 2014), and PPIX has been shown to catalyze the conversion of hydrogen peroxide into singlet oxygen species (Zeng et al 2015). Work with RDT at higher MV energies that are more clinically relevant is ongoing, although initial studies have been promising, with 5-ALA combined with 15-MV or 45-MV radiotherapy (RT) in single and multi-fractionated treatments being shown to enhance cell killing compared to RT alone using both in vitro and in vivo models (Ma et al 2014, Wang et al 2015, Zhang et al 2016).

The objective of this study was to investigate the synergistic effect of a novel treatment consisting of high-energy (15-MV) radiotherapy combined with 5-aminolevulinic acid (5-ALA) as a photosensitizer prodrug, and carbamide peroxide to generate oxygen species, using an in vivo mouse model. The combined effect of the photosensitizers and high-energy RT on treatment outcome is thus compared to the effect of the individual components. To the authors’ knowledge, this is the first work to investigate the effects of this particular combination of treatments used for radiodynamic therapy, as well as the first to study radiodynamic therapy with a small animal model using 15-MV radiation.

2. Methods and materials

2.1. Tumor perfusion

To verify that blood perfusion in the tumors was adequate to deliver the 5-ALA, an 18 F-FDG PET/CT study was performed using a G8 preclinical scanner (Sofie BioSciences, Inc. Culver City, CA) on a subset (N = 10) of the mice used for the in vivo study. Imaging was performed weekly, with each mouse administered ∼80 μCi of 18 F-FDG retro-orbitally, with a rest period of 1 h between injection and scanning. Analysis of the images was performed using VivoQuant (inviCRO, Boston, MA) post-processing software. Contouring was performed using a thresholding algorithm used in other investigations (Ford et al 2006, Lee 2010, Werner-Wasik et al 2012). The threshold value for each image was given by:

Threshold=f(M−B)+B, (1)

where f is a fixed percentage, M is the maximum voxel value, and B is the background voxel value, determined by averaging the voxels within the area immediately surrounding the tumor (minimum volume = 9 cm3). As the PET/CT images were used qualitatively for these studies, a single threshold value of 40% was chosen for each image, as this value led to reasonable contours, and is close to the optimal value for accurate volume measurement for spheres close to the size of the tumors in these images (Davis et al 2006, Drever et al 2006).

2.2. In vivo study

The mouse model, cell line, and injection technique were similar to that described in (Wang et al 2019); in brief, the mouse model used was the male athymic BALB/c at six weeks old, purchased from Harlan (Indianapolis, IN) in groups of 25–50 for each experiment. The cell line studied in this work was a human prostate cancer line (PC-3) from American Type Culture Collection (Manassas, VA), cultured in RPMI-1640.

Tumors consisted of 3 × 106 tumor cells given in 100 μl of Dulbecco’s phosphate-buffered saline (PBS), and were injected subcutaneously into the flanks of the mice; tumors were injected bilaterally in order to increase the number of tumors studied. 1–2 weeks was given to allow the tumors to grow at least to a size at which they could be visualized on MR (average volume = 180 mm3). All mice were kept at room temperature and given a 12-hour day/night schedule, given access to food and water freely, and euthanized ∼2 weeks after treatment.

In total, the tumors were randomized into 8 groups. The treatment groups included an untreated (control) group and various combinations of RT, 5-ALA, and carbamide peroxide in order to determine the individual and synergistic effects of the treatment components: 1. Untreated, 2. RT, 3. 5-ALA, 4. Carbamide peroxide, 5. RT + 5-ALA, 6. RT + carbamide peroxide, 7. 5-ALA + carbamide peroxide, 8. RDT (RT + 5-ALA + carbamide peroxide).

Radiation was delivered using a Varian Clinac iX (Varian Medical Systems, Palo Alto, CA). 15-MV radiation was used, since this was the highest energy available and production of Cerenkov radiation increases with the speed of the secondary electrons (Tamm and Frank 1937, Axelsson et al 2011). Mice were treated in groups of 2–3 after anesthesia with a ketamine solution, and were positioned supine on the RT treatment couch with 2.5 cm bolus (Action Products, Inc. Hagerstown, MD). Radiation was delivered at a gantry angle of zero degrees and at a source-to-surface distance (SSD) of 100 cm, with the treatment consisting of a single dose of approximately 4 Gy localized to the two tumors with a 10 × 3 cm2 collimator opening. 5-ALA was administered by tail-vein injection ∼4 h prior to RT, to allow for the drug to distribute throughout the body. The determination of the timing was chosen based on our assumption that 4 h after drug administration, there would be reasonable drug concentration in the tumor volume through blood circulation (Stummer et al 1998, Yamamoto et al 2015), based in part on prior experience. Carbamide peroxide was used to deliver hydrogen peroxide to tissue, and was administered intratumorally into the tumors ∼3–5 min before RT; the determination of this timing was based on our assumption that RT would be delivered before significant metabolism or reaction of the peroxide in vivo. All injections and procedures were performed using methods approved by the institutional animal care and use committee.

2.3. Chemicals

5-ALA (Cayman Chemicals, Ann Arbor, MI) was diluted in PBS, and the final 5-ALA solution was delivered at a concentration of 100 mg kg−1. Carbamide peroxide (Sigma Aldrich, St. Louis, MO) was diluted in PBS, and delivered at a concentration of 60 mg kg−1. The ketamine solution for anesthesia consisted of 60 mg kg−1 of ketamine (VEDCO Inc. Saint Joseph, MO), mixed with 2.5 mg kg−1 of ace-promazine (Boehringer Ingelheim, Ridgefield, CT), with ∼15 μl delivered by intramuscular injection per sedation.

2.4. Tumor volume measurement

Tumor growth was monitored with weekly caliper measurements, as well as weekly MR scans using a GE 1.5 T Signa MR scanner (GE Healthcare, Waukesha, WI) and a 3-in diameter surface coil, on which 1–2 mice were imaged per scan. MR images were acquired using the FSE T2-weighted sequence (TR/TE = 2200/85 ms), resulting in a resolution of 0.243 × 0.243 × 1.2 mm3 per voxel and a field of view of 7 × 7 cm2 for axial scans.

Tumor volume was measured with the calipers by measuring two perpendicular lengths of the tumor and using a formula found elsewhere in the literature (Takahashi et al 2013):

Tumorvolume=1/2d2D, (2)

where d is the shorter diameter and D is the larger diameter. Tumor volume was measured from the MR scans by contouring each slice of the axial scans on a program developed within our institution. Tumors were contoured used Canny edge detection to define tumor boundaries. Tumor growth for each tumor was quantified as the ratio of tumor volume one and two weeks after treatment to that immediately before treatment:

TumorGrowth=TumorVolume(t~7/14days)/TumorVolume(t~0days), (3)

2.5. Statistical analysis

The Student’s t-test was used to compare the RDT group individually to the RT only, RT + 5-ALA, and RT + carbamide peroxide groups, while Welch’s ANOVA test was used to compare the RT only, RT + 5-ALA, and RT + carbamide groups to determine any statistically significant difference in tumor growth among the groups. The Kruskal-Wallis test was used to compare the control, carbamide peroxide only, 5-ALA only, and 5-ALA + carbamide peroxide groups, since the sample sizes of these groups were small and the data were not sufficiently similar to normal distributions. The Student’s t test was additionally used to compare growth for several treatment groups after dividing each into two groups, according to initial tumor size. A p value <0.05 was considered to be significant. Note that data for the various treatment groups were acquired over several experiments; in order to control for variations in the growth of the PC-3 cells among different batches of cells, data for the various treatment groups used for the statistical analyses were collected only from relevant experiments in which the treatment groups being compared were included.

3. Results

Representative 18F-FDG PET/CT images, with and without threshold contouring, for one mouse (figures 1(a)–(c)) show that tumors are clearly visualized and that 18 F-FDG is therefore present within the tumors. Comparison to the MR image, with edge detection implemented, of the same mouse (figure 1(d)) shows that for some of the tumors imaged in this study, regions of the tumor that appear solid on the MR scans are not included in the threshold-based contouring algorithm on the PET/CT scans, indicating these regions are not as metabolically active or as well perfused as other regions. Blood perfusion is therefore shown to be adequate to deliver agents to the tumors involved in this study, although they may be delivered non-uniformly, depending on the tumor.

Figure 1.

Figure 1.

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Comparison of MR and 18 F-FDG PET/CT images for one mouse, showing that perfusion is adequate for tumors to uptake 18 F-FDG and that uptake is uneven for tumors that appear solid on MR images. (a): 3D rendering of a PET/CT image, with dotted line indicating the slice at which MR and PET/CT transaxial images are taken. (b) Transaxial PET/CT, (c): Transaxial PET/CT with threshold contouring, (d): Transaxial MR with edges delineated.

Figure 2 shows a plot of tumor volume measurements made with MR compared to those made using calipers for >450 tumors measured in this study. Measurements made with MR increase with those made using calipers with a linear fit of slope 1.049 ± 0.054, consistent with the expected slope of 1, and therefore lending confidence in the methodology of MR measurement used in this study. Because of the inherently better precision of the MR scans, all tumor growth calculations reported in this paper were made using the MR measurements.

Figure 2.

Figure 2.

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Comparison of tumor volume measured using MR images to that measured using calipers, along with linear fit, y = (1.049 ± 0.054)x + (73 ± 27).

RT led to a decrease in tumor growth relative to the control group (39.4 ± 4.9% at two weeks post-treatment), as expected (figure 3). RDT resulted in a significant decrease in tumor growth compared to RT (24 ± 9% and 21 ± 8% at one and two weeks post-treatment, respectively), while the carbamide peroxide + RT group and 5-ALA + RT group did not show statistically decreased tumor growth relative to the RT group (figure 4(a)). Additionally, the carbamide peroxide only, 5-ALA only, and 5-ALA + carbamide peroxide groups did not show statistically decreased tumor growth compared to the control group (figure 4(b)), indicating that peroxide and 5-ALA contribute significantly to tumor growth delay only when administered together with RT.

Figure 3.

Figure 3.

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Tumor growth curve, showing average tumor volume normalized to the volume on the treatment day, for tumors in the control (N = 64) and RT only (N = 49) groups at one and two weeks after treatment. All measured tumor values were used for these measurements, and all error bars show the standard error of the mean; *p < 0.05.

Figure 4.

Figure 4.

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Upper Left: Tumor growth curves normalized to the volume of the RT group at two weeks after treatment, fo the RT onlly (N = 26), RT + 5-ALA (N = 26), RT + Carb. Per. (N = 22), and RDT (N = 27) groups. Upper Right: Tumor growth curves normalized to the volumen of the control group at two weeks after treatment. for the Control (N = 12), Carb. Per. (N = 16), and 5-ALA only/5-ALA. + Carb. Per. (N = 6) groups. Bottom Left: Average tumor volume at two weeks after treatment, normalized to the volumet on the treatment day. for tumors in the treatment groups treated with radiation. RDT resulted in. a greater tumor-growth delay relative to RT, RT + Carb. Per, and RT + 5-ALA. *p < 0.05; **p < 0.02. Bottom Right: Average tumor volume at two weeks after treatment, normalized to the volume on the treatment day, for tumors in the treatment groups treated without radiation. All error bars show the standard error of the mean.

Figure 5 compares tumor growth for several of the treatment groups when the tumors were divided according to initial tumor size into two groups; a tumor volume of 180 mm3 was chosen to divide the tumor groups since this led to an adequate number of tumors in each group. The treatment groups show a statistically insignificant difference in tumor growth among the tumor size groups, and the trend of decreased tumor growth with the various treatments is maintained among the tumor size groups.

Figure 5.

Figure 5.

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Top: Tumor growth curves for tumors divided according to initial tumor size (minimum N = 9). Bottom: Tumor growth ratio at two weeks post-treatment for tumors divided according to initial tumor size. Treatment growth is not statistically different for the two tumor size groups for each treatment group (*p > 0.05). All error bars show the standard error of the mean.

4. Discussion

18F-FDG PET/CT scans indicated that some tumors have regions that are not metabolically active or are poorly perfused, potentially leading to a lesser effect for treatments that rely on delivery of chemicals through the vasculature. While a more detailed analysis of the metabolic growth of the tumors was outside the scope of this study, these data suggest that metabolic imaging may clarify the volume of the tumors that are treated. The comparison of tumor volume measured with MR to that measured with calipers verified that the two methods of tumor measurement used in this study were sensibly correlated. Without a ground truth against which to compare, it did not fully validate the use of MR to measure tumor volume. Nevertheless, previous investigations (Montelius et al 2012) have shown that MR is suitable to accurately measure volume and is preferred over caliper measurements. While MR measurements are inherently more costly and time-consuming than caliper measurements, MR-based tumor volume determination was particularly useful for the irregularly shaped tumors involved in this study.

RDT was shown to slow tumor growth relative to RT alone, and was additionally shown to produce an effect greater than the effect of the individual constituents. Furthermore, RDT was shown to have an effect on tumor growth delay for each of the tumor size groups into which the data were sorted. The results therefore demonstrate the potential of using clinically relevant, high radiation energies in radiodynamic therapy, and warrant an investigation into the impact of the radiation energy on the effectiveness of the treatment. Moreover, the effect produced by the RDT designed in this study highlights the benefit of combining an oxidizing agent to the photosensitizer and radiation in this treatment; further systematic studies are necessary in order to determine the mechanism by which the peroxide acts in concert with 5-ALA for the treatment and the optimal dose/fractionation scheme for future clinical applications. While the dosage and timing of both 5-ALA and carbamide peroxide have been shown to affect tumor growth, these have not yet been optimized in these initial experiments, and the reduction in tumor growth by RDT by 57% relative to the control demonstrated in this study has significant therapeutic potential. Clinical photosensitizers are known to absorb preferentially in the visible light range; however, the mechanism of action by which x-rays activate these photosensitizers is still the subject of investigation, leaving uncertain the optimal methodology and parameters with which to deliver this treatment (Takahashi and Iwahashi 2017). Additionally, radiation was delivered in a single fraction in this study in order to determine the effect on tumor growth of a single dose of radiation. Tumor growth delay measured in the experiments could therefore more directly relate to cell killing from each treatment delivery. Experiments are warranted to study the effect of RDT delivered daily over multiple fractions in order to increase the tumoricidal effect and incorporate other cellular mechanisms of radiation treatment (e.g. cellular repair).

5. Conclusions

Radiodynamic therapy consisting of 15-MV radiotherapy combined with 5-ALA and carbamide peroxide resulted in a more significant tumor growth delay compared to the individual constituents in this initial study. RDT led to a decrease in tumor growth of 24 ± 9% and 21 ± 8% at one and two weeks post-treatment, respectively, relative to RT alone. The possibility of radiodynamic therapy using high clinical radiation energies, along with the combined effect of the photosensitizing agent and an oxidizing agent in radiodynamic therapy, has therefore been demonstrated.

Acknowledgments

This publication was supported by Grant No. P30 CA006927 from the National Cancer Institute, NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health. We would like to thank the core research facilities at Fox Chase Cancer Center for their technical support.

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