Carbon Ion Irradiation Suppresses Tumor Growth, Migration and Invasion in X-ray Resistant Melanoma in Vitro and in Vivo

Abstract

Objectives

To investigate whether carbon ion irradiation (CIR) can overcome the inherent radioresistance of melanoma and suppress metastasis, we evaluated its effects on B16F10 cells in vitro and in vivo.

Methods

B16F10 cells and C57BL/6J tumor-bearing mice were irradiated with either 4-MV X-rays or 290 MeV/nucleon carbon ions at different doses. Metastatic ability was assessed by wound-healing, Transwell migration, and Matrigel invasion assays. Expression of metastasis-related proteins, including MMP2, MMP9, N-cadherin, and E-cadherin, was analyzed by Western blotting or immunohistochemistry (IHC). Tumor growth and survival were recorded, and H&E staining was performed for histopathological evaluation.

Results

Under identical physical-dose conditions, CIR suppressed cell migration and invasion more effectively than XIR, especially at 2.5 and 7.5 Gy. These effects were accompanied by reduced expression of MMP2, MMP9, and N-cadherin, along with increased E-cadherin expression. Furthermore, 7.5 Gy CIR significantly suppressed tumor growth, reduced tumor volume, and prolonged survival in tumor-bearing mice, although tumor regrowth was observed after 21 days. Conversely, 0.5 Gy XIR exerted promoting effects, while 2.5 Gy XIR only had a slight impact.

Conclusion

These findings highlight the antimetastatic potential of CIRT against radioresistant melanoma. However, further validation with RBE-adjusted doses, human cell models, and metastasis-related endpoints is warranted.

Graphical Abstract.

Introduction

Malignant melanoma (MM), a tumor arising from melanocytes (the pigment-producing cells of the skin), remains a highly lethal malignancy. ¹ As the most aggressive form of skin cancer, melanoma has seen its incidence rise faster than that of any other cancer over the past five decades. ² In the United States alone, an estimated 104,960 new cases of invasive melanoma were diagnosed in 2025. ³ Unlike most cancers, melanoma incidence is not strongly dependent on age; it is among the leading causes of cancer and cancer-related mortality in young adults aged 20-35 years. ⁴ In Australia, MM is the eighth leading cause of cancer-related death, ⁵ while in the United States, it is the second most frequently diagnosed cancer among adolescents and young adults (aged 15-29 years).^6,7 By disproportionately afflicting young and middle-aged populations, melanoma imposes substantial socioeconomic and psychological burdens.

Metastasis is the primary cause of melanoma-related mortality. ⁸ Owing to its pronounced propensity for dissemination and rapid metastatic progression, metastatic melanoma has historically been associated with a dire prognosis and has been largely incurable. Conventional chemotherapy has demonstrated limited efficacy, and traditional adoptive cell therapies have been applicable to only a small subset of patients. Consequently, prior to the advent of modern systemic treatments, fewer than 10% of patients with advanced disease survived beyond a few years. ⁹ Although recent advances in targeted therapies and immunotherapies have improved outcomes for some patients with metastatic disease,^10,11 the development of treatment resistance remains a major obstacle in MM management.^9,12 Therefore, there is an urgent need to identify more effective preventive and therapeutic strategies.

With the development of imaging and radiation delivery techniques, radiotherapy has become an important adjuvant therapy for advanced or metastatic melanoma. ¹³ However, the inherent radioresistance of melanoma cells has largely restricted its use to postoperative adjuvant settings or to patients who are not candidates for surgery. ¹⁴ Moreover, several studies suggest that conventional low-linear energy transfer (LET) photons (e.g., X- and γ-rays) administered at sublethal or clinically relevant doses may paradoxically enhance the metastatic potential of tumor cells.^15-17 In contrast, high LET carbon ion beams have demonstrated promising clinical outcomes in the treatment of MM.^18,19 Published studies further indicated that carbon ion beams exert antimetastatic effects both in vitro and in vivo,^20-22 suggesting superior metastasis suppression compared with low-LET photon radiation. Nevertheless, the metastatic potential of tumor cells following irradiation is determined not solely by the radiation modality but also by the intrinsic biological properties.^23,24 Our previous work has shown that carbon ion IR (CIR) induces a higher rate of cell death, and a more pronounced and sustained G2/M arrest than X-rays. ²⁵ Building on these findings, the present study aims to investigate the effects of CIR on the metastatic behavior of B16F10 models both in vitro and in vivo.

Materials and Methods

Cell Culture

Murine B16F10 melanoma cells were obtained from the National Collection of Authenticated Cell Culture (Shanghai, China). Cells were cultured in RPMI-1640 medium (Gibco Life Technologies, Carlsbad, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Hyclone, GE Healthcare Life Sciences, Logan, USA) and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific Inc.) at 37°C in a humidified 5% CO₂ atmosphere. For irradiation experiments, cells were seeded into 25 cm² culture flasks (Corning, 430168) at a density optimized for exponential growth and were irradiated with either X-rays or carbon ion beams at room temperature.

Mice and Subcutaneous Tumor Model

Female C57BL/6J mice (aged 6-8 weeks) were obtained from the Laboratory Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences. All mice were housed under specific pathogen-free (SPF) conditions in the animal facility of the Institute of Modern Physics (IMP), with free access to food and water and 12-h light/dark cycle. To assess the tumor growth inhibition and metastasis-related protein expression, each mouse received a subcutaneous injection of 2 × 10⁶ B16F10 cells suspended in 100 μL of phosphate-buffered saline (PBS) into the right flank. When tumor growth reached a diameter of 7-9 mm, mice were randomly divided into 7 groups (n=10 per group): non-irradiated control, X-ray irradiation (XIR) at 0.5, 2.5, 7.5 Gy, and CIR at 0.5, 2.5, 7.5 Gy. Irradiation was performed with a single fraction at the corresponding dose. Tumor volume was measured every other day using a caliper and calculated as V = 0.5 × length × width². Body weight and survival were recorded on the same schedule. At the experimental endpoint, tumors were harvested, fixed, and embedded in paraffin for pathological analysis. All animal procedures were conducted in accordance with the guidelines approved by Animal Care and Use Committee of IMP.

Irradiation

CIR was performed at the Heavy Ion Research Facility (HIRF) in Lanzhou (using an energy of 290 MeV/nucleon and a LET of 75 keV/μm). To ensure that the target volume was fully encompassed within the spread-out Bragg peak (SOBP), a 167 mm solid water phantom was positioned in the beam path field above the tumor. XIR was carried out using a small-animal irradiator (4 MV, 225 kV and 13.3 mA) at a dose rate of 1.0 Gy/min. A lead collimator was used to shield the surrounding tissue and confine radiation exposure to the tumor region. Dose verification was performed using an ionization chamber that was calibrated by the National Institute of Metrology, China.

Wound-Healing Assay

Wound-healing (scratch) assay was performed to evaluate the migratory capacity of B16F10 cells in vitro. Briefly, 3 × 10⁵ cells were seeded into 60 mm culture dishes and cultured in RPMI 1640 medium containing 10% FBS until they reached approximately 90% confluence. The cells were then serum-starved for 24 h to synchronize cell cycle progression. After irradiation with graded doses of carbon ions or X-rays, a linear scratch was introduced across the cell monolayer using a sterile 200 µL pipette tip. Cellular debris was removed by washing twice with PBS, and fresh medium containing 10% FBS was added. Images of the scratch were captured at 0 h and 24 h post-irradiation using an inverted microscope (Nikon Eclipse Ti-S, Japan). Wound closure was quantified by measuring the remaining scratch area using ImageJ software. For each dish, three randomly selected fields were analyzed, and all experiments were performed independently in triplicate.

Migration Assay

Cell migration was further evaluated using 6.5 mm diameter Transwell^® inserts (Corning, NY, USA) with 8 µm pore polycarbonate membranes in a 24-well plate. Briefly, irradiated B16F10 cells were washed three times with PBS, trypsinized, and resuspended in serum-free RPMI 1640 medium. 5×10⁴ cells in 200 µL RPMI 1640 containing 5% FBS were seeded into the upper chamber. The lower chamber was filled with 500 µL RPMI 1640 with 20% FBS to generate a chemotactic gradient. After 24 h of incubation at 37°C in 5% CO₂, non-migrating cells on the upper surface of the membrane were carefully removed with a cotton swab. Cells that had migrated to the lower surface were fixed in 4% paraformaldehyde for 30 min and stained with 0.01% crystal violet (Sigma-Aldrich) for 15 min at room temperature. After rinsing with distilled water, stained cells were photographed and counted in five randomly selected fields per well using an inverted microscope (Axio observer, Carl Zeiss, Germany). All experiments were performed in triplicate.

Invasion Assay

Cell invasive ability was assayed using the same Transwell® system, except that the polycarbonate membranes were pre-coated with 20 µL of Matrigel (Corning, NY, USA) diluted 1:8 in serum-free medium. Irradiated cells (5×10⁴) were seeded, incubated, fixed, stained, and visualized following the same protocol as described for the migration assay. All experiments were performed independently in triplicate.

Western Blotting

Irradiated B16F10 cells were lysed on ice in RIPA lysis buffer (Beyotime, Shanghai, China) containing 1% PMSF. Lysates were centrifuged at 12,000 × g for 15 min at 4°C, and supernatants were collected for protein quantification. Total protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo, USA). Equal amounts of protein (20 µg per lane) were mixed with 5× SDS loading buffer, boiled for 5 min, and separated on 10% SDS-PAGE gels, and electro-transferred onto PVDF membranes (Millipore, IPVH00010). Membranes were blocked with 5% bovine serum albumin (BSA, BBI Life Sciences Corporation, Canada) in Tris-buffered saline containing 0.05% Tween-20 (TBST) for 2 hours at room temperature. Then the membranes were incubated overnight at 4°C with primary antibodies against MMP-2 (1:200, ab92536), MMP-9 (1:1000, ab58803), E-cadherin (1:2000, ab40772), N-cadherin (1:800, ab7601), and β-actin (1:5000, AP0060). After three washes with TBST, the membranes were incubated with appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using an electrochemiluminescence detection system (Millipore, Darmstadt, Germany). Protein bands were captured with AI680 imaging system (Alpha Innotech Corporation) and quantified using ImageQuant TL software (version 8.1; GE Healthcare Life Sciences). All target protein bands were normalized to β-actin, and relative expression levels were calculated as fold change versus the non-irradiated control.

Histopathological Assay

Morphological changes were evaluated by hematoxylin-eosin (H&E) stained sections. Tumor tissues were collected at the experimental endpoint, fixed in 10% neutral buffered formalin for 24 h, embedded in paraffin, and sectioned at 4 μm thickness using a microtome (Leica RM2235, Germany). Sections were deparaffinized in xylene, rehydrated through a graded ethanol series, and stained with hematoxylin and eosin (H&E). Morphological changes, including necrosis, cavitation, and cellular atypia, were examined under a light microscope (Nikon Eclipse Ci-L, Japan) by a pathologist blinded to the treatment groups.

Immunohistochemical (IHC) Assay

IHC staining was carried out using a two-step polymer detection system (PV-9000, ZSBG-Bio) following the manufacturer’s recommended protocol. Briefly, sections were deparaffinized, rehydrated, and subjected to antigen retrieval by boiling in 10 mM sodium citrate buffer (pH 6.0) for 15 min in a microwave oven. Endogenous peroxidase was blocked by incubation with 3% H₂O₂ in methanol for 15 min at room temperature. Non-specific binding was minimized by blocking with 10% normal goat serum for 30 min. The sections were then incubated overnight at 4°C with primary antibodies against MMP-2 (ab86607; 1:200), MMP-9 (ab283575; 1:1000), E-cadherin (ab40772; 1:8000), and N-cadherin (ab7601; 1:2000). After three washes with PBS (5 min each), sections were incubated with secondary antibodies (anti-mouse/anti-goat IgG) for 1 h. Immunoreactivity was visualized using 3,3′-diaminobenzidine (DAB, ZLI-9018, ZSBG-Bio) as the chromogen, and sections were counterstained with hematoxylin. For quantification, ten randomly selected high-power fields (200× magnification) were examined per section, and the percentage of positively stained tumor cells was scored by two independent observers who were blinded to the experimental groups.

Statistical Analysis

All data are presented as the mean ± standard deviation (SD). A two-tailed Student’s t-test was used for pairwise comparison between two independent groups. Two-way analysis of variance (ANOVA) followed by Tukey’s post hoc multiple comparison test was performed for multi-group comparisons with two variables. Survival curves were constructed using the Kaplan–Meier method, and log-rank tests were utilized to evaluate intergroup survival differences. A p-value of less than 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism (version 8.0, San Diego, CA, USA).

All cell experiments were conducted between October 2020 and August 2022, and animal experiments were conducted between March 2024 and November 2025.

Results

CIR Reduces B16F10 Cell Migration in Vitro

To determine whether CIR affects melanoma cell migration, we performed wound-healing and Transwell migration assays following exposure to different doses of X-rays or carbon ion beams. As shown in Figures 1 and 2, low-dose X-rays (XIR-0.5) significantly increased cell migration relative to non-irradiated controls, while CIR produced a dose-dependent suppression of migration. Of note, cells treated with 7.5 Gy CIR showed 45% less scratch closure than those receiving 7.5 Gy X-rays (Figure 1A and B), indicating superior inhibitory efficacy of CIR at the same physical dose. Similar to scratch results, low-dose XIR (0.5Gy) stimulated more cells to migrate. Notably, CIR at 7.5 Gy reduced the number of migrated cells by nearly 80% compared to the non-IR group (Figure 2A and B). Collectively, these findings demonstrate that CIR exerts a significantly stronger anti-migratory effect on B16F10 cells than conventional XIR.

Figure 1.

CIR inhibits scratch closure more effectively than XIR in B16F10 cells. (A) Representative scratch images at 0 and 24 h (×100). (B) Quantification of wound closure at 24 h post irradiation. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group; #p < 0.05, ##p < 0.01, ###p ≤ 0.001 vs. XIR group

Figure 2.

CIR reduces B16F10 cell migration more effectively than XIR. (A) Representative images of migrated cells at 24 h post-irradiation (×200). (B) Quantification of migrated cells. A Transwell migration assay was used. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group; #p < 0.05, ##p < 0.01, ###p≤ 0.001 vs. XIR group

CIR Suppresses B16F10 Cell Invasion in Vitro

To further evaluate the anti-metastatic potential of CIR, we examined the invasive capacity of B16F10 cells following exposure to different doses of X-rays or carbon ions using Matrigel-coated Transwell chambers. As shown in Figure 3, low-dose XIR (0.5 Gy) also stimulated invasive potential, with the number of invading cells increasing by approximately 10% compared to the non-irradiated control. In contrast, CIR at 2.5 and 7.5 Gy significantly reduced cell invasion, decreasing the number of invading cells by about 63% and 82%, respectively (Figure 3A and B). Taken together, these results demonstrate that CIR exerts a potent inhibitory effect on the invasive capacity of radioresistant B16F10 cells, further supporting its anti-metastatic potential.

Figure 3.

CIR suppresses B16F10 cell invasion more effectively than XIR. (A) Representative images of invaded cells at 48 h post-irradiation (×200). (B) Quantification of invaded cells. Matrigel-coated Transwell chambers were used. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group; #p < 0.05, ##p < 0.01, ###p< 0.001 vs. XIR group

CIR Downregulates Metastasis-Related Protein Expression in Vitro

To elucidate the molecular mechanisms underlying the inhibitory effects of CIR on B16F10 cell migration and invasion, we examined the expression of metastasis-associated proteins by Western blotting. As presented in Figure 4, MMP-2 and MMP-9 expression was significantly increased following XIR at 0.5 and 2.5 Gy, whereas XIR at 7.5 Gy and CIR at 2.5 and 7.5 Gy led to a marked reduction in their expression. Notably, CIR induced a more substantial decrease in MMP-2 and MMP-9 expression than XIR at the same physical doses (Figure 4A–C). In terms of epithelial-mesenchymal transition (EMT) markers, E-cadherin expression was suppressed by XIR at 0.5 and 2.5 Gy but elevated at 7.5Gy, while N-cadherin showed the opposite trend. Compared with XIR, CIR exerted a stronger effect on both E-cadherin induction and N-cadherin suppression (Figure 4D–F). Collectively, these findings indicate that CIR suppresses B16F10 cell migration and invasion, likely through modulating the expression of key proteins involved in extracellular matrix (ECM) degradation and EMT.

Figure 4.

CIR more effectively suppresses metastasis-related protein expression in B16F10 cells. (A) Representative Western blots and (B, C) quantitative densitometric analysis of MMP-2 and MMP-9 expression. (D) Representative Western blots and (E, F) quantitative densitometric analysis of the E cadherin and N-cadherin expression. *p < 0.05, **p < 0.01, ***p< 0.001 vs. control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. XIR group

CIR Suppresses Tumor Growth and Prolongs Survival in Tumor-Bearing Mice

To further evaluate the antitumor and antimetastatic efficacy of CIR in vivo, we established a subcutaneous B16F10 cell-derived tumor model in C57BL/6J mice. When tumors reached 7-9 mm in diameter, mice were treated with different doses of CIR or XIR. As shown in Figure 5, CIR at 7.5 Gy effectively suppressed tumor growth, with sustained reduction in tumor size and volume for over 21 days. In contrast, although 7.5 Gy XIR and 2.5 Gy CIR also slowed tumor progression, rapid regrowth occurred within two weeks after irradiation (Figure 5C). Of note, survival analysis showed that mice receiving 7.5 Gy XIR or CIR at 2.5 or 7.5 Gy exhibited a significant survival benefit compared with the non-irradiated control (Figure 5D). These results indicate that high-dose CIR not only effectively controls local tumor growth but also confers a substantial survival advantage.

Figure 5.

CIR suppresses melanoma tumor growth more effectively than XIR in C57BL/6J mice. (A) Representative images of tumor size, and (B) Tumor volume at 14 days after irradiation. (C) Time-dependent tumor growth curves following treatment with different doses of XIR or CIR. (D) Kaplan-Meier survival curves of tumor-bearing C57BL/6J mice over 28 days after irradiation. **p < 0.01, ***p < 0.001 vs. non-IR control group

CIR Induces Tumor Cell Necrosis and Cavitation in Vivo

To assess the histological changes induced by XIR or CIR, we performed H&E staining on tumor sections collected at the experimental endpoint. As presented in Figure 6, both 7.5 Gy XIR and CIR induced pronounced tumor cell necrosis and cavitation, characterized by extensive loss of cellular architecture and formation of cystic spaces. Notably, tumors treated with 7.5 Gy CIR exhibited the most severe cavitation and nearly complete tumor elimination, whereas the effects of XIR were less extensive. These histopathological findings provide direct evidence that CIR exerts a stronger cytotoxic effect against B16F10 cells than conventional XIR in vivo.

Figure 6.

Histological changes in B16F10 melanoma tumors following CIR or XIR. H&E staining, ×200

CIR Downregulates Metastasis-Related Protein Expression in Vivo

To further corroborate the findings in vitro, we examined the expression of key metastasis-associated proteins in tumor tissues by IHC. As presented in Figure 7A and B, MMP2 and MMP9 expression was substantially reduced by 7.5 Gy XIR or CIR at both 2.5 and 7.5 Gy. In contrast, 0.5 Gy XIR or CIR significantly upregulated MMP2 and MMP9 expression. With respect to EMT markers, E-cadherin and N-cadherin expression exhibited no significant alterations following either XIR or CIR. However, 7.5 Gy CIR induced a modest increase in E-cadherin and a corresponding decrease in N-cadherin (Figure 7C and D), suggesting a partial shift toward a less aggressive phenotype. Collectively, these in vivo results are consistent with the in vitro data, further supporting the anti-metastatic potential of CIR.

Figure 7.

IHC analysis of metastasis-related protein expression in B16F10 melanoma tumors following treatment with different doses of XIR or CIR. Representative images of tumor sections stained with specific antibodies against MMP-2, MMP-9, E-cadherin, and N-cadherin, visualized with 3,3′-diaminobenzidine (DAB, brown) and counterstained with hematoxylin (blue), ×200

Discussion

Malignant melanoma (MM) is a cutaneous malignancy originating from melanocytes. ²⁶ Its incidence has been increasing rapidly worldwide, posing a growing public health concern. ³ Although melanoma accounts for only about 5% of all skin cancers, it is responsible for the majority of skin cancer-related deaths. ²⁷ Early-stage melanoma can be cured with surgical excision, but a considerable proportion of patients still face a poor prognosis and high mortality due to its strong invasion and metastasis ability.^14,28 Current therapeutic strategies for MM include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. ²⁸ For patients with unresectable or advanced disease, adjuvant therapies represent a critical treatment option.^14,28,29 With the advancement in imaging and radiotherapy (RT) techniques, RT has become an important adjuvant modality in the multidisciplinary management of advanced or metastatic MM. ¹³ Nevertheless, the intrinsic radioresistance of melanoma cells largely limits the application of conventional photon-based radiotherapy, whether used as postoperative adjuvant therapy or as a primary treatment for inoperable patients. In this context, therapeutic approaches that can improve both tumor control and suppression of metastatic behavior are of considerable interest.

Carbon ion radiotherapy (CIRT) exhibits favorable clinical outcomes owing to its distinct physical and biological advantages, such as higher relative biological effectiveness (RBE) and superior dose conformity, compared with conventional photon-based radiotherapy.^18,30 However, despite promising local control, CIRT has not consistently achieved satisfactory 5-year survival rates in certain cancers, including MM.^14,31 Distant metastasis remains a major determinant of mortality in solid tumors, suggesting that enhanced systemic control could improve long-term survival in CIRT-treated patients. Although CIRT is considered a promising treatment approach for MM, available preclinical and clinical data remain limited and occasionally contradictory. Several published studies have indicated that CIR exerts stronger cytotoxic effects on melanoma cells than low-LET photon irradiation. For instance, Aninditha KP et al demonstrated that ¹² C and ¹⁶ O heavy ions more effectively suppressed proliferation and elicited greater G2/M phase arrest than XIR in melanoma cells. ³² Similarly, Saito K et al reported superior cell-killing efficacy of carbon ions relative to γ-rays. ³³ Matsumoto Y et al observed that even low-LET CIR more potently suppressed metastatic formation in B16/BL6 cells than XIR, whereas low-dose X-rays (0.5 Gy) promoted metastases. ²¹ More recently, Charalampopoulou et al reported that photon and carbon ion irradiation induced radiation-type-dependent phenotypic changes in human vaginal mucosal melanoma cells, including dendrite formation, pigmentation, and alterations in migration and invasion behavior. ²² These findings indicate that irradiation may alter melanoma cell behavior in ways that are not limited to direct cytotoxicity. In our previous study, we observed that CIR induced more pronounced and sustained cytotoxicity in B16F10 melanoma cells than XIR, with an RBE of approximately 3.5. ²⁵ In the present study, we further demonstrated that CIR significantly suppresses migration (Figures 1 and 2) and invasion (Figure 3) in X-ray-resistant B16F10 cells. In vivo, CIR induced extensive tumor cell necrosis and cavitation (Figure 6), inhibits tumor growth, and prolongs survival (Figure 5). The findings of the present study are in agreement with previous reports in lung^21,34,35 and pancreatic cancers,^36,37 reinforcing that CIR is more effective than XIR, not only in killing tumor cells but also in suppressing metastasis in MM.

Nonetheless, while these data provide compelling evidence for the enhanced efficacy of CIR in B16F10 cells and tumor-bearing mice, a critical caveat must be acknowledged: the present study compared CIR and XIR at equal physical doses without RBE adjustment. Given that carbon ions have a higher RBE than photons (typically 2-3, depending on endpoint), the observed superiority of CIR may partly reflect its greater biological effectiveness rather than LET-specific effects alone. This distinction matters because RBE is endpoint-dependent, higher for DNA damage and cell killing, but potentially lower for migration suppression or immunomodulation. Future studies with RBE-normalized doses are needed to disentangle these contributions.

In addition, tumor regrowth was observed in the present study at ∼21 days post-CIR, which indicates that a single CIR treatment is insufficient for durable tumor eradication. This recurrence may reflect the persistence of a viable radioresistant fraction after irradiation. One plausible contributor is the survival of stem-like melanoma cells, which have been linked to tumor heterogeneity, metastasis, and long-term tumor growth.^38,39 This observation supports future evaluation of fractionated or repeated CIR, as well as combination strategies with chemotherapy, targeted therapy, or immunotherapy, to improve durable tumor control. For instance, Sai S et al demonstrated that combination of CIR and cisplatin exerted higher growth-inhibitory effects than CIR alone in xenografted malignant pleural mesothelioma. ⁴⁰

Metastasis is a multistep process in which malignant cells migrate from the primary tumor to discrete organs via the bloodstream or lymph vessels. During this process, the extracellular matrix (ECM) poses a major barrier that must be degraded. MMP-2 and MMP-9 facilitate cell migration by playing a pivotal role in ECM degradation.^41-43 Accumulating evidence indicates that overexpression of MMP-2 and MMP-9 is closely associated with invasion and metastasis in MM. For instance, El-Shabrawi Y et al reported a positive correlation between MMP-2 and MMP-9 in MM. ⁴⁴ Another study showed MMP-9 promotes melanoma invasion and metastasis. ⁴⁵ Moreover, elevated MMP-2 and MMP-9 in the tumor microenvironment can directly drive epithelial-mesenchymal transition (EMT). It has been indicated that MMPs degrade E-cadherin, a key epithelial marker in EMT, leading to disruption of the cell-cell adhesion. ⁴⁶ Normally, cells are held together by tight and adherent junctions, which are formed by E-cadherin. Loss of E-cadherin along with elevation of N-cadherin is associated with tumor progression, metastasis, and poor prognosis.^47,48 In the present study, we observed that CIR more effectively suppressed MMP-2 and MMP-9 expression, subsequently elevated E-cadherin, and downregulated N-cadherin in B16F10 cells (Figure 4) and tumor (Figure 7) than XIR. These findings suggest that, compared with XIR, CIR more effectively suppresses migration and invasion phenotypes by regulating the expression of metastasis-related proteins.

Although this study was limited to a single murine melanoma cell line and a subcutaneous tumor model, which do not fully recapitulate the biological heterogeneity or metastatic progression of human melanoma, the present data nevertheless suggest that CIR is more effective than XIR in suppressing aggressive phenotypes in B16F10 melanoma. Further studies incorporating additional models, biologically equivalent dose comparisons, and combination strategies with systemic therapies are warranted to better define the translational potential of carbon ion radiotherapy in melanoma management.

Conclusion

In summary, the present study demonstrates that, under identical physical-dose conditions, CIR exerts superior antitumor activity compared with XIR, as evidenced by more potent suppression of B16F10 cell migration and invasion in vitro and delayed tumor growth in vivo. Notably, while low-dose XIR (0.5 Gy) appeared to promote migration- and invasion-associated phenotypes, CIR consistently suppressed these behaviors across all tested doses, accompanied by downregulation of MMP2 and MMP9 expression. However, tumor regrowth observed at approximately 21 days after CIR treatment indicates that a single dose of carbon ion irradiation is insufficient to achieve durable tumor eradication. This observation, together with the regrowth data, suggests that to optimize therapeutic outcomes in melanoma metastasis, combination approaches integrating CIR with chemotherapy, targeted therapy, or immunotherapy warrant further investigation.

ORCID iD

Aihong Mao https://orcid.org/0000-0002-2927-8976