Recently, modern therapies involving immune checkpoint inhibitors, cytokines, and oncolytic virus have been developed. Because of the limited treatment effect of modern therapy alone, the immunostimulatory effect of radiotherapy attracted increasing attention. The combined use of radiotherapy and modern therapy has been examined clinically and non-clinically, and its effectiveness has been confirmed recently. Because melanomas have high immunogenicity, better therapeutic outcomes are desired when using immunotherapy. However, sufficient therapeutic effects have not yet been achieved. Thus far, radiotherapy has been used only for local control of tumors. Although extremely rare, radiotherapy has also been reported for systemic control, i.e., abscopal effect. This is thought to be due to an antitumor immune response. Therefore, we herein summarize past information on not only the mechanism of immune effects on radiotherapy but also biomarkers reported in case reports on abscopal effects. We also reviewed the animal model suitable for evaluating abscopal effects. These results pave the way for further basic research or clinical studies on new treatment methods for melanoma. Currently, palliative radiation is administered to patients with metastatic melanoma for local control. If it is feasible to provide both systemic and local control, the treatment benefit for the patients is very large.

Chemo-radiotherapy, which combines chemotherapy with radiotherapy, has been clinically practiced since the 1970s, and various anticancer drugs have been shown to have a synergistic effect when used in combination with radiotherapy. In particular, cisplatin (CDDP), which is often the cornerstone of multi-drug combination cancer therapies, is highly versatile and frequently used in combination with radiotherapy for the treatment of many cancers. Therefore, the mechanisms underlying the synergistic effect of CDDP and radiotherapy have been widely investigated, although no definitive conclusions have been reached. We present a review of the combined use of CDDP and radiotherapy, including the latest findings, and propose a mechanism that could explain their synergistic effects. Our hypothesis involves the concepts of overlap and complementation. “Overlap” refers to the overlapping reactions of CDDP and radiation-induced excessive oxidative loading, which lead to accumulating damage to cell components, mostly within the cytoplasm. “Complementation” refers to the complementary functions of CDDP and radiation that lead to DNA damage, primarily in the nucleus. In fact, the two concepts are inseparable, but conceptualizing them separately will help us understand the mechanism underlying the synergism between radiation therapy and other anticancer drugs, and help us to design future radiosensitizers.

Melanoma is a highly aggressive cancer with a propensity for brain metastases. These can be treated by radiotherapy, but the radiation-resistant nature of melanoma makes the prognosis for melanoma patients with brain metastases poor. Previously, we demonstrated that treatment of mice with subcutaneous melanoma with 5-aminolevurinic acid (5-ALA) and X-rays in combination, (“radiodynamic therapy (RDT)”), instead of with 5-ALA and laser beams (“photodynamic therapy”), improved tumor suppression in vivo. Here, using the B16-Luc melanoma brain metastasis model, we demonstrate that 5-ALA RDT effectively treats brain metastasis. We also studied how 5-ALA RDT damages cells in vitro using a B16 melanoma culture. Cell culture preincubated with 5-ALA alone increased intracellular photosensitizer protoporphyrin IX. On X-ray irradiation, the cells enhanced their ∙OH radical generation, which subsequently induced γH2AX, a marker of DNA double-strand breaks in their nuclei, but decreased mitochondrial membrane potential. After two days, the cell cycle was arrested. When 5-ALA RDT was applied to the brain melanoma metastasis model in vivo, suppression of tumor growth was indicated. Therapeutic efficacy in melanoma treatment has recently been improved by molecular targeted drugs and immune checkpoint inhibitors. Treatment with these drugs is now expected to be combined with 5-ALA RDT to further improve therapeutic efficacy.

Recent reports have suggested that 5-aminolevulinic acid (5-ALA), which is a precursor to protoporphyrin IX (PpIX), leads to selective accumulation of PpIX in tumor cells and acts as a radiation sensitizer in vitro and in vivo in mouse models of melanoma, glioma, and colon cancer. In this study, we investigated the effect of PpIX under X-ray irradiation through ROS generation and DNA damage. ROS generation by the interaction between PpIX and X-ray was evaluated by two kinds of probes, 3′-(p-aminophenyl) fluorescein (APF) for hydroxyl radical (•OH) detection and dihydroethidium (DHE) for superoxide (O₂^•-). •OH showed an increase, regardless of the dissolved oxygen. Meanwhile, the increase in O₂^•- was proportional to the dissolved oxygen. Strand breaks (SBs) of DNA molecule were evaluated by gel electrophoresis, and the enhancement of SBs was observed by PpIX treatment. We also studied the effect of PpIX for DNA damage in cells by X-ray irradiation using a B16 melanoma culture. X-ray irradiation induced γH2AX, DNA double-strand breaks (DSBs) in the context of chromatin, and affected cell survival. Since PpIX can enhance ROS generation even in a hypoxic state and induce DNA damage, combined radiotherapy treatment with 5-ALA is expected to improve therapeutic efficacy for radioresistant tumors.

To treat malignant glioma, standard fractionated radiotherapy (RT; 60 Gy/30 fractions over 6 weeks) was performed post-surgery in combination with temozolomide to improve overall survival. Malignant glioblastoma recurrence rate is extremely high, and most recurrent tumors originate from the excision cavity in the high-dose irradiation region. In our previous study, protoporphyrin IX physicochemically enhanced reactive oxygen species generation by ionizing radiation and combined treatment with 5-aminolevulinic acid (5-ALA) and ionizing radiation, while radiodynamic therapy (RDT) improved tumor growth suppression in vivo in a melanoma mouse model. We examined the effect of 5-ALA RDT on the standard fractionated RT protocol using U251MG- or U87MG-bearing mice. 5-ALA was orally administered at 60 or 120 mg/kg, 4 h prior to irradiation. In both models, combined treatment with 5-ALA slowed tumor progression and promoted regression compared to treatment with ionizing radiation alone. The standard fractionated RT protocol of 60 Gy in 30 fractions with oral administration of 120 and 240 mg/kg 5-ALA, the human equivalent dose of photodynamic diagnosis, revealed no significant increase in toxicity to normal skin or brain tissue compared to ionizing radiation alone. Thus, RDT is expected to enhance RT treatment of glioblastoma without severe toxicity under clinically feasible conditions.

Introduction: 5-Aminolevulinic acid (5-ALA)-based radiodynamic therapy (RDT), an experimental approach that combines systemic administration of 5-ALA with ionizing radiation, has demonstrated antitumor efficacy primarily in preclinical studies using fractionated irradiation protocols. In recent years, single-dose and hypofractionated irradiation regimens have been increasingly adopted in clinical radiotherapy; however, the therapeutic potential of 5-ALA-based RDT under single-dose irradiation conditions remains to be elucidated. Methods: We evaluated the accumulation of protoporphyrin IX (PpIX) in human colon cancer HT-29 cells compared with previously studied cell lines, mouse melanoma B16/BL6 and human glioblastoma U-251 MG, U-87 MG in vitro . Using a HT-29 xenograft mouse model, we investigated the antitumor efficacy of a single 12 Gy X-ray dose combined with 5-ALA at doses of 100 or 200 mg/kg. Tumor growth, histopathological alterations, and immune cell infiltration were analyzed. Gene expression profiles of tumor tissues were examined by microarray analysis at day 29 post-irradiation. Results: HT-29 cells exhibited equal or greater PpIX accumulation compared with other tumor cell lines. In vivo , single-dose X-ray irradiation (12 Gy) combined with various doses of 5-ALA resulted in 5-ALA dose-dependent suppression of tumor growth. Notably, administration of 200 mg/kg 5-ALA plus 12 Gy X-ray induced marked tumor regression in all animals without statistically significant weight loss. Histopathological analysis demonstrated disruption of tumor cell islands and increased infiltration and proximity of Iba1-positive immune cells to tumor cells. Microarray analysis identified 75 differentially expressed genes between untreated and 200 mg/kg 5-ALA plus X-ray groups, including downregulation of genes involved in DNA repair, tumor suppression, autophagy, cell cycle regulation, metabolism, and immune evasion. Conclusion: This study demonstrates for the first time that 5-ALA combined with single-dose X-ray irradiation exerts a strong antitumor effect on HT-29 xenografts. The observed effects may be mediated by induction of immunogenic cell death, modulation of the tumor microenvironment, and suppression of tumor cell survival pathways. These findings highlight single-dose 5-ALA-based RDT as a potential novel therapeutic strategy.