Abstract
Background/Aim: The antitumor effects of 5-aminolevulinic acid (5-ALA) radiodynamic therapy (RDT) have been demonstrated in vitro and in vivo in several malignancies. However, its application in bone and soft tissue sarcomas has not been sufficiently explored. In this study, we aimed to elucidate the efficacy of 5-ALA RDT in osteosarcoma cell lines.
Materials and Methods: In vitro studies utilized human (143B) and mouse (LM8) osteosarcoma cells. Cultured cells were either untreated (control) or exposed to 5-ALA (0-1,000 μg/ml) followed by X-ray irradiation (a single dose of 5 Gy). Cell viability was measured 48 h post-irradiation. Both 143B and LM8 cells were inoculated subcutaneously into the backs of BALB/c mice. Mice were assigned to untreated (control), Rx (irradiation alone), or 5-ALA RDT groups. Mice in the Rx group received X-ray irradiation (5 Gy) after macroscopic tumor formation, while those in the 5-ALA RDT group received 5-ALA (250 mg/kg) intraperitoneally, followed by 5 Gy X-ray irradiation. The mice were sacrificed 14 days after treatment, and changes in tumor volume were evaluated.
Results: In vitro, 5-ALA RDT treatment of osteosarcoma cells at 200, 500 and 1,000 μg/ml significantly inhibited cell proliferation at 48 h post-treatment, compared to control cells. In mice injected with osteosarcoma cells, the 5-ALA-RDT group showed a significant reduction in tumor volume compared to the control and Rx groups. No significant changes in body weight or abnormal behavior were observed.
Conclusion: 5-ALA RDT demonstrated significant anti-tumor effects in both in vitro and in vivo osteosarcoma models. These findings suggest its potential as a therapeutic approach for osteosarcoma.
Introduction
Osteosarcoma (OS) is the most common primary bone sarcoma affecting children and adolescents (1). The survival of patients with non-metastatic OS has improved considerably since the 1970s owing to the establishment of treatment with surgical resection and perioperative chemotherapy (2, 3). However, the five-year survival rate remains at 60-80% over 30 years (2, 3). The treatment regimens of doxorubicin, cisplatin, methotrexate, and ifosfamide have not changed over the last three decades, thereby necessitating the development of new therapeutic strategies for further improvement of survival in patients with OS (2-4).
5-Aminolevulinic acid (5-ALA), a natural amino acid and a precursor of protoporphyrin IX (PpIX), is a photo- and radiosensitizer produced during the mitochondrial heme biosynthesis pathway (5, 6). PpIX exhibits several biological activities. It selectively accumulates in cancer cells as a photo- and radiosensitizer due to the low activity of ferrochelatase (FECH), an enzyme responsible for metabolizing PpIX into haem (5, 6). 5-ALA photodynamic diagnosis (PDD) has been widely used in recent years for the diagnosis of brain tumors and bladder cancer. Tumor tissues specifically emit red fluorescence at approximately 635 nm after 5-ALA administration with blue light excitation at 405 nm (7-9). After light irradiation, photosensitizers, such as PpIX, generate reactive oxygen species (ROS) in the presence of oxygen, and increased ROS levels can induce apoptosis and necrosis of targeted cancer cells (10, 11). PpIX has also been reported to be a radiosensitizer. Following irradiation, PpIX exerts anti-tumor effects through ROS generation (12-14).
The efficacy of 5-ALA radiodynamic therapy (RDT) has been demonstrated in vitro and in vivo in several malignancies, including brain cancer, prostate cancer, malignant melanoma, lymphoma, and lung cancer (15-21). However, its application in bone and soft tissue sarcomas has not been sufficiently explored. Therefore, the present study aimed to evaluate the anti-tumor effects of the 5-ALA RDT on LM8 and 143 B osteosarcoma cell lines in vitro and in vivo.
Materials and Methods
Osteosarcoma cell lines and culture. LM8 (murine osteosarcoma, Suita, Osaka, Japan) and 143B (human osteosarcoma, Riken Cell Bank, Ibaraki, Japan) cell lines were cultured in minimum essential medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO2.
In vitro studies. Cells were divided into two groups: control and 5-ALA RDT. Cells were seeded in 96-well plates at a density of 1×104 cells per well in 50 μl of medium and cultured for 24 h. The control cells were not treated with 5-ALA or irradiated. Cells in the 5-ALA RDT group were treated with 50 μl of 5-ALA solution (FUJIFILM WAKO Chemicals Corporation, Kanagawa, Japan) to achieve final concentrations of 0, 200, 500, or 1,000 μg/ml. After a one-hour incubation, the cells were exposed to a single 5 Gy dose of X-rays (150 kV, 20 mA). Cell viability was assessed using the Cell Titre 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA) at 0, 16, 24, and 48 h post-irradiation.
In vivo studies. Five-week-old male BALB/c mice were subcutaneously inoculated on the backs with LM8 or 143B cells (2×106 cells suspended in 200 μl PBS). When the tumor diameter exceeded 10 mm, mice were assigned to one of the three groups: untreated control, irradiation (Rx group; single dose of 5 Gy), and 5-ALA RDT (intraperitoneal administration of 5-ALA at 250 mg/kg followed by a single dose of 5 Gy irradiation). The mice in the 5-ALA-RDT and irradiation groups were anaesthetized using a combination of anesthetics (0.75 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol). The actual adjusted volume of the anesthetics was 1.875 ml of medetomidine, 2 ml of midazolam, 2.5 ml of butorphanol, and 18.625 ml of physiological saline, for a total of 25 ml. Tumor dimensions were measured using callipers, and both body weight (g) and tumor volume (mm3) were recorded twice weekly. The mice were observed for two weeks, after which they were sacrificed. Tumor volume was calculated as follows: V=π × (major axis) × (minor axis)2/6 (22).
Statistical analysis. All in vitro experiments were repeated at least three times to ensure reproducibility. Data are expressed as mean±standard deviation (SD). Differences between the two groups were analyzed using the Mann–Whitney U-test. In addition, a one-way analysis of variance (ANOVA) was performed, followed by Tukey’s multiple comparison test. Statistical significance was set at p<0.05. All statistical analyses were performed using EZR (Saitama Medical Centre, Jichi Medical University, Saitama, Japan), a graphical user interface for R (R Foundation for Statistical Computing, Vienna, Austria), a modified version of R Commander that incorporates statistical functions commonly used in biostatistics.
Ethics. All animal experiments were conducted according to the principles and guidelines for the care and use of laboratory animals in research, testing, and education. The study protocol was approved by the Ethics Committee of the authors’ affiliated institutions (approval number 2022-12). We have adhered to the ARRIVE guidelines and have supplied the ARRIVE Checklist.
Results
Antitumor efficacy of 5-ALA RDT in LM8 and 143B osteosarcoma cell lines. The antitumor effects of 5-ALA RDT were evaluated in LM8 and 143 B cells treated with 0-1,000 μg/ml of 5-ALA. LM8 cells treated with 200, 500, and 1,000 μg/ml of 5-ALA showed a significant reduction in cell viability as early as 16 h compared to untreated (0 μg/ml of 5-ALA) cells (p<0.01) (Figure 1A). This inhibitory effect was significantly higher at 24 h and 48 h (p<0.01). Compared to the control group, the Rx (5-ALA 0 μg/ml) and 5-ALA RDT groups showed significant decreases in cell proliferation (p<0.01) (Figure 1A).
Effect of 5-Aminolevulinic acid (5-ALA) and radiodynamic therapy (RDT) on cell proliferation. (A) LM8 and (B) 143B osteosarcoma cells were treated with 5-ALA (0-1,000 μg/ml). After a one-hour incubation, the cells were exposed to a single 5 Gy dose of X-rays. Cell viability at each time point was compared with that at 0 h. Asterisks indicate significant differences between groups (*p<0.01).
Similarly, in 143B cells, 5-ALA RDT induced a significant reduction in cell viability as early as 16 h at concentrations of 200, 500, and 1,000 μg/ml, compared to untreated (5-ALA 0 μg/ml) cells (p<0.01). The inhibition was time-dependent with significantly higher decreases at 24 h and 48 h (p<0.01) (Figure 1B). Control (untreated and unirradiated) and untreated (5-ALA 0 μg/ml) cells in the 5-ALA RDT group showed no difference in cell proliferation.
5-ALA administration and RDT in the xenograft model. On day 14 post-irradiation, none of the groups had any dead mice, and all mice were included in this study. In the LM8 model, no apparent differences in body weight were observed between the control, Rx, and 5-ALA RDT groups (n=4 per group) during the 14-day observation period. Compared to the control group, the Rx group showed a significant reduction in tumor volume. In addition, the tumor volume was significantly lower in the 5-ALA RDT group compared to both the control and Rx groups (Figure 2A).
Effect of 5-Aminolevulinic acid (5-ALA) and radiodynamic therapy (RDT) on tumor growth in a xenograft osteosarcoma model. When the tumor diameter exceeded 10 mm, mice were either left untreated (control) or treated with a single dose of irradiation (5 Gy, Rx group) or 5-ALA (250 mg/kg) followed by a single dose of irradiation (5 Gy, 5-ALA RDT group). Mice were sacrificed 14 days after treatment, and tumor volumes were measured using callipers. Asterisks indicate significant differences between groups (*p<0.05, **p<0.01).
On day 14, the mean relative tumor volume ratio of the control group was 9.35 [standard deviation (SD)=5.19, range=4.22-15.87], whereas those of the Rx and 5-ALA RDT groups were 2.93 (SD=0.34, range=2.69-3.44, p=0.0286) and 1.31 (SD=0.20, range=1.15-1.60, p=0.0286), respectively. A statistically significant difference was observed between the Rx and 5-ALA RDT groups (p=0.0286).
In the 143 B model, there were no apparent differences in body weight between the control, Rx, and 5-ALA RDT groups (n=5 in each group) during the 14-day observation period. Compared to the control group, the Rx group showed a significant reduction in tumor volume. In addition, the tumor volume was significantly lower in the 5-ALA RDT group compared to both the control and Rx groups (Figure 2B).
On day 14, the mean relative tumor volume ratio of the control group was 4.57 (SD=2.03, range=2.66-7.85), whereas those of the Rx and 5-ALA RDT groups were 2.20 (SD=0.40, range=1.89-2.89, p=0.0159) and 1.76 (SD=0.15, range=1.58-1.89, p=0.00794), respectively. A statistically significant difference was observed between the Rx and 5-ALA RDT groups (p=0.00794).
Discussion
In this study, we elucidated the efficacy of the 5-ALA RDT in osteosarcoma cell lines, both in vitro and in vivo. Interestingly, 5-ALA-RDT treatment was more effective than radiotherapy alone in inhibiting tumor growth in an osteosarcoma xenograft model.
When administered systemically, 5-ALA is taken up by tumor cells, where the decreased activity of FECH results in the selective accumulation of PpIX within the mitochondria (5, 6). In previous studies, Adachi et al. demonstrated lower expression of FECH following 5-ALA PDT treatment in LM8 and 143 B cells, as well as in HT1080 fibrosarcoma cells, compared to that in C2C12 mouse myoblast cells, using a western blot assay (23). FECH plays a crucial role in the regulation of PpIX levels and exerts anti-tumor effects through irradiation-induced ROS, which cause various types of cellular damage and disrupt mitochondrial function (12-14).
The efficacy of 5-ALA RDT has been reported in several types of cancers in vivo and in vitro (Table I) (15-21). In particular, in vivo studies in brain, prostate, lung, and colorectal cancer cells have shown significant inhibition of tumor growth after 5-ALA RDT treatment (Table I). Additionally, this study for the first time demonstrates the efficacy of 5-ALA RDT in an osteosarcoma xenograft model.
Recent in vivo studies (past 10 years) of 5-ALA RDT.
Radiotherapy is not routinely used for osteosarcoma (24-26). In clinical practice, it is administered as a local control measure for inoperable or incompletely resected osteosarcoma (24-26). While osteosarcoma is known for its relative resistance to photon radiotherapy, such as X-ray radiotherapy, there are few reports on the effectiveness of photon therapy (27). More recently, radiosensitizers have been a new focus in clinical research (25, 28). Adachi et al. demonstrated the efficacy of 5-ALA photodynamic therapy (PDT) in osteosarcoma and fibrosarcoma cell lines, both in vitro and in vivo (23). However, 5-ALA PDT can only be applied to superficial tumors and the surgical field due to the limited penetration depth of the light. Based on the findings of the present study, we believe that 5-ALA RDT can overcome this limitation and can be applied to deep tumors, such as unresected osteosarcomas of the spine or pelvis.
In the clinical setting, the single use of 5-ALA PDD and PDT for cancers such as brain and bladder tumors is well-established (7-9, 29-32). However, no studies have evaluated the safety and efficacy of repeated oral 5-ALA applications during multiple fractions of radiotherapy, although this has been evaluated in several in vivo studies (15-18, 20, 21). In Germany, a Phase I/II study is ongoing for the first-in-human evaluation of repeated oral administration of 5-ALA as a radiosensitizer for the treatment of recurrent glioblastoma (33).
A limitation of this study is the lack of power calculation and reporting of side effects of the present treatment. Further research is warranted to address these limitations and confirm these findings. Furthermore, treatment protocols for osteosarcoma, with optimal timing and dose of irradiation, should be evaluated in an osteosarcoma model in a preclinical setting.
In conclusion, this study demonstrated the anti-tumor efficacy of 5-ALA RDT in both in vitro and in vivo models using the LM8 and 143 B osteosarcoma cell lines. Compared to radiation therapy alone, 5-ALA RDT results in a significantly stronger inhibitory effect on tumor growth. These findings suggest that the 5-ALA RDT has the potential to become a clinically applicable treatment modality for osteosarcoma.
Footnotes
Authors’ Contributions
Tomoki Nakamura: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resource, Data curation, Writing-original draft, Visualization, Project administration, Funding acquisition. Yuki Tanemura: Methodology, Software, Resource, Data curation, Formal analysis, Investigation, Writing-original draft. Yumi Matsuyama: Validation, Resource, Data curation. Tomohito Hagi: Formal analysis, Resource, Investigation, Takahiro Iino: Project administration, Resource, Data curation, Supervision. Masahiro Hasegawa: Project administration, Funding acquisition, Supervision, Writing-editing & review.
Conflicts of Interest
The Authors declare no conflicts of interest in the present study.
Funding
This work was supported by JSPS KAKENHI Grant Number JP23K08629.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received March 31, 2026.
- Revision received April 20, 2026.
- Accepted April 24, 2026.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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