Cancer remains one of the leading threats to human health and is associated with a reduction in life expectancy. Studies have shown that the prevalence of cancer and mortality rates are rising worldwide, as per a recent report from the Global Cancer Report, which indicated that approximately 14 million new cases were diagnosed in 2022 1. Additionally, the report projected that cancer cases will increase by up to 60% over the next 20 years 2. Advances in early detection, screening, diagnosis, and treatment have contributed to a modest decline in cancer mortality 3; however, comprehensive worldwide cancer data indicate that further research is needed to reduce cancer mortality 3,4. Cancer treatment involves a variety of approaches, including surgery, radiation therapy, chemotherapy, immunotherapy, targeted therapy, stem cell transplantation, and multidisciplinary strategies 5.

Radiotherapy (RT) primarily induces DNA damage, leading to activation of the DNA damage response (DDR). The intricate DDR pathway maintains genome stability by activating proteins responsible for detecting, signaling, and transmitting damage signals to effector proteins that regulate cell cycle progression, arrest, DNA repair, and apoptosis; this is one of the biological effects of ionizing radiation (IR) on normal cell function 6. Over half of cancer patients undergo RT, with at least 40% experiencing clinical benefits. However, treatment resistance remains a significant obstacle that reduces the effectiveness of radiotherapy 7. Various signaling pathways linked to tumour development have provided deeper insights into cancer biology, leading to the development of new targeted therapies. Multiple signaling pathways, including the phosphoinositide 3-kinase (PI3K/AKT), JAK/STAT, transforming growth factor beta (TGFβ), Wnt, and NF-κB signalling pathways, are often interconnected in cancer research. Numerous studies have found that alterations in the PI3K/AKT pathway are commonly linked to cellular transformation, carcinogenesis, cancer development, and treatment resistance 8. In our previous study, we reported an increase in Ctla4 and Pdcd1 expression, which may contribute to acquired radioresistance in an in vitro model via the PI3K/AKT and JAK/STAT pathways 9. Research has shown that the PI3K/AKT signalling pathway is frequently overactivated in cancer cells resistant to radiation, chemotherapy, and hormonal therapy 10. Additionally, evidence suggests that dual targeting of PI3K and mTOR may reduce radiation resistance in various cancer cell types, both in vitro and in vivo xenograft models 11.

The PI3K/AKT signalling pathway has been proposed to play a key role in the development of radiotherapy resistance, making it a promising target for further investigation. This pathway regulates several hallmarks of cancer, including cell survival, metastasis, and metabolism. It is also involved in tumour microenvironment remodeling, affecting angiogenesis and recruitment of inflammatory cells 12. A variety of chromosomal alterations, such as mutations in PIK3CA, phosphatase and tensin homolog (PTEN), AKT, TSC1, and mechanistic target of rapamycin (mTOR), can lead to abnormal activation of the PI3K/AKT pathway 13. The PI3K/AKT pathway is frequently mutated and activated in cancer 14.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is another critical signalling pathway involved in cellular responses to cytokines and growth factors 15. The JAK/STAT pathway has been identified as mediating resistance to radiotherapy in various cancers 16. While acquired resistance arises from activation of alternative signalling pathways, de novo resistance results from genetic changes in receptors or downstream signalling molecules in the JAK2/STAT3 pathway 17. Previous studies have highlighted the JAK/STAT pathway as a crucial mediator of radioresistance 16.

The PI3K/AKT and JAK/STAT pathways play important roles in mediating cellular responses to radiation and immune checkpoint blockade. Crosstalk between these pathways and CTLA-4 signaling can influence tumour radioresistance and immune evasion, thereby impacting the efficacy of radiation therapy and immunotherapy. Understanding the interplay between these pathways is crucial for developing effective therapeutic strategies to overcome treatment resistance and improve patient outcomes in cancer.

Building on our previous in vitro findings (Sham et al., 2023), which reported increased PD-1 and CTLA-4 expression in the radioresistant EMT6^RR_MJI cell line, the present study extends this work into an in vivo setting to examine whether similar immune-checkpoint modulation occurs in the whole-tumour microenvironment. Although the EMT6^RR_MJI model shares features with the existing BALB/c–EMT6 models, it represents a stably acquired, fractionation-induced radioresistant phenotype, offering an opportunity to compare immune-regulatory responses between resistant and non-resistant tumours. By assessing radiation-associated changes in immune-checkpoint expression in this more physiologically relevant context, the study provides additional insight into how tumour–immune interactions may influence radioresistance and could inform future strategies combining radiotherapy with immunomodulatory approaches.

This study extends our previous in vitro observations (Sham et al., 2023) by confirming, for the first time in vivo, that Pdcd1 and Ctla-4 are differentially regulated in radioresistant versus parental EMT6 tumours following gamma-ray exposure. While prior murine breast cancer models of radioresistance have focused largely on DNA repair, apoptosis, and signalling pathways, our findings introduce a novel immunoregulatory dimension to the mechanism of radioresistance. The divergent modulation of PD-1 expression between resistant and non-resistant tumours indicates that immune checkpoint adaptation may represent a key distinguishing feature of the radioresistant phenotype. This not only enhances mechanistic understanding but also identifies PD-1 as a potential biomarker and therapeutic target in resistant breast cancer.

Furthermore, by integrating checkpoint profiling with tumour growth analysis, this study establishes a comprehensive in vivo platform for evaluating radio-immunomodulatory interactions—an aspect largely absents from prior murine breast cancer models. The work therefore provides both conceptual and methodological novelty that extends beyond our previous in vitro research and contributes meaningfully to the evolving framework of radio-immunobiology.

This study did not include an a priori power analysis, and the use of six mice per group may have reduced the power to detect small to moderate effects, particularly in gene expression analyses where inter-individual variability can be substantial. The findings should therefore be interpreted with caution, and validation using larger sample sizes and appropriately powered experimental designs is recommended for future work.

Cancer metastasis remains a leading cause of cancer-related death, with primary tumour cells spreading by infiltrating blood vessels, invading the surrounding microenvironment, and migrating to distant organs to form secondary tumours 22. A key process driving metastasis in many epithelial cancers is the epithelial-to-mesenchymal transition (EMT), where cancer cells undergo genetic reprogramming, transforming from a non-motile, epithelial phenotype to a more migratory, mesenchymal-like phenotype. This transformation enhances the tumour’s malignancy and invasiveness 23. A hallmark of EMT is the downregulation of epithelial cadherin gene (Cdh1), and upregulation of neural cadherin gene (Cdh2) 24. E-cadherin and N-cadherin proteins are calcium-dependent cell adhesion molecules that regulate cell–cell adhesion and migration and tumour invasiveness. Loss of E-cadherin–mediated adhesion plays a vital role in the progression of epithelial tumours from benign to invasive forms 25. Successful creation of the xenograft model using EMT6 cells was confirmed analysing the expression of the Cdh1 and Cdh2 epithelial markers. Cdh2 was upregulated, whereas the Cdh1 was downregulated. Cdh2 serves as a marker for mesenchymal cells 26, whereas Cdh1 is a marker for epithelial cells 26. Therefore, the overexpression of Cdh2 and downregulation of Cdh1 support the idea that the radioresistant tumour originated from EMT6 cells.

PD-1 proteins and its ligand, PD-L1 which are part of the immunoglobulin superfamily, serve as crucial inhibitory checkpoint proteins that regulate T cell signalling. In resting immune cells, Pdcd1 expression is low 27. However, Pdcd1 overexpression helps tumour cells evade cytotoxic T lymphocytes and the development of anti-PD-1/PD-L1 antibodies has become a central focus of cancer immunotherapy 28. Overexpression of Cd274 mRNA has been shown to predict clinical outcomes in patients with low-grade glioma following radiotherapy 29, and its role in immune response modulation is further evidenced by the development of autoimmune diseases in mice lacking Pdcd1 expression. Among breast cancer patients, a significant proportion (55-59%) exhibit overexpression of Pdcd1/ Cd274 mRNA 30. According to Chen et al. (2016), PD-L1 activates an inhibitory signalling pathway that prevents T cell activation 31. This blocked immune-mediated cell death allows tumour cells to proliferate and survive within the tumour microenvironment 32 contributing to treatment resistance. PD-1 and PD-L1, as well as the CTLA-4 immune checkpoint pathways help maintain peripheral tolerance by reducing T cell activation. Cancer cells utilize these pathways to create an immunosuppressive environment, enabling tumour growth and proliferation rather than immune system destruction 33. These findings align with these current studies, which showed increased Pdcd1 and Cd274 expression in the EMT6^RR_MJI tumour model.

CTLA-4 is a cell-surface receptor protein that inhibits T cells from transmitting immunological signals 34. It functions alongside regulatory T cells (Tregs) as part of a complementary, overlapping immune tolerance mechanisms. The suppressive activity of Tregs is reduced when Ctla4 mRNA expression is impaired 35,36. CTLA-4 competes with its homolog CD28 for binding to the ligands CD80/CD86, thereby interfering with CD28-mediated T cell activation and decreasing the immune response 37. Overexpression of Ctla4 in four different breast cancers and tumorigenic cell lines suggests its role in resistance to radiotherapy 38. Accordingly, Ctla4 overexpression has been linked to poor survival outcomes in patients with breast cancer 38. Interestingly, a better prognosis has been associated with Ctla4 overexpression in tumour-infiltrating lymphocytes (TILs), highlighting the critical role of CTLA-4 protein in helping tumours escape immune responses 30,39.

Radioresistance in cancer cells can be affected by various mechanisms, including reoxygenation, DNA repair, apoptosis, and proliferation 40. The current study employed a fractionated radiation dose to allow tumour reoxygenation between fractions, thereby increasing tumour sensitivity to radiotherapy 41. The results indicated that mice with radioresistant EMT6 cells (EMT6^RR_MJI) were unaffected by irradiation, whereas parental EMT6 tumours undergoing fractionated irradiation showed growth regression. A previous study showed that higher radiation doses with fewer fractions (10 Gy/ 2 fraction per week) could reduce tumour growth, increase anti-tumour immunoreactivity, and limit delayed radio-necrosis 42. Accordingly, mesenchymal stem cells restrict growth and promote apoptosis by inhibiting the proliferation of cancer cells 43. Mesenchymal cells also improve the effects of radiotherapy on malignancies, most likely by reducing tumour cell proliferation and enhancing cancer cell death 44. Taken together, the regression of tumours in the treated parental EMT6 cell model was due to (i) higher fractional irradiation doses that increased tumour control and immunoreactivity, as well as (ii) the presence of mesenchymal cells in the tumour.

The contrasting responses between parental EMT6 and EMT6^RR_MJI cells suggest potential mechanisms like post-radiotherapy hypoxia and early acquisition of radioresistance in the latter 41. A previous study reported that the effectiveness of radiotherapy in hypoxic tumours decreases as cancer cells adapt to hypoxic conditions and become more resistant to radiation 45. Tumour cells may escape the radiation effects by migrating and penetrating the vessels. Although this study did not measure hypoxic markers, it has been shown that irradiation can cause hypoxia in tumours which hampers immune cells and causes immune suppression and poor prognosis after radiation therapy 46. Irradiation induces radioresistance in cancer cells of EMT tumours through multiple signalling pathways and the tumour microenvironment (TME) 47. Fractionated irradiation can upregulate Cd274 mRNA expression and cause changes in the tumour microenvironment 48. This is consistent with the results of the present study, where EMT6^RR_MJI tumour cells showed increased Pdcd1 and Cd274 expression, resulting in increased tumour growth after irradiation. Targeting these markers might enhance the radiosensitivity in tumour tissue. Another factor is the presence and increase of EMT cells in tumours, which promotes cancer-associated fibroblast formation in tumours. Radiation-induced mesenchymal transition can lead to the abnormal recruitment of pericytes in the tumour vasculature during tumour regrowth after radiotherapy 49. Radiation-induced mesenchymal transition, observed in EMT6^RR_MJI cells, contributes to acquired radioresistance, emphasizing the importance of inhibiting this process to enhance radiotherapy efficacy, mainly through immune response promotion. 50.

Pdcd1 expression was elevated in the acquired radioresistant EMT6^RR_MJI cells, with gamma-ray irradiation resulting in a reduction of Pdcd1 levels. In contrast, Pdcd1 and Ctla4 expression were lower in parental EMT6 cells but increased following irradiation. Gamma-ray irradiation did not significantly affect tumour volume in the EMT6^RR_MJI model, likely reflecting the inherent resistance of these cells. Overall, this study provides preliminary evidence suggesting that radiation may modulate immune checkpoint expression differently in resistant and non-resistant tumour models. These findings highlight potential interactions between immune regulation, radiation response, and tumour microenvironment dynamics that warrant further mechanistic and functional investigation.

AKT: Protein kinase B; ANOVA: Analysis of variance; ARRIVE: Animal Research: Reporting of In Vivo Experiments; ATCC: American Type Culture Collection; BALB/c: Bagg Albino laboratory-bred mouse strain; cDNA: Complementary DNA; CD28: Cluster of differentiation 28; CD80/CD86: Cluster of differentiation 80/86; CTLA-4: Cytotoxic T-lymphocyte-associated protein 4; DDR: DNA damage response; DMEM: Dulbecco's Modified Eagle Medium; DNA: Deoxyribonucleic acid; EMT: Epithelial-to-mesenchymal transition; EMT6^RR_MJI: Acquired radioresistant EMT6 subline; FBS: Fetal bovine serum; FIG: Figure; Gy: Gray (unit of radiation dose); IR: Ionizing radiation; JAK: Janus kinase; LAFAM: Laboratory Animal Facility; mRNA: Messenger RNA; mTOR: Mechanistic target of rapamycin; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; PD-1: Programmed cell death protein 1; PD-L1: Programmed death-ligand 1; PI3K: Phosphoinositide 3-kinase; PTEN: Phosphatase and tensin homolog; qPCR: Quantitative polymerase chain reaction; REC: Research Ethics Committee; RNA: Ribonucleic acid; RT: Radiotherapy; STAT: Signal transducer and activator of transcription; TGFβ: Transforming growth factor beta; TILs: Tumour-infiltrating lymphocytes; TME: Tumour microenvironment; Tregs: Regulatory T cells; UiTM: Universiti Teknologi MARA

The study was partly funded by the Fundamental Research Grant Scheme, Ministry of Education Malaysia (FRGS/1/2019/SKK08/UITM/02/9) and Lestari Research Grant, Universiti Teknologi MARA (600-RMC/MYRA 5/3/LESTARI (094/2020)).

Funding acquisition: MJI, NAH, HHH

Conception: NRFS, MJI

Methodology: NFRS, NAH, MJI

Interpretation or analysis of data: NRFS, NAHH, MJI

Preparation of the manuscript: NH, NAHH, NH, WNIWMZ, MKAK, SBSAF, SO, NJO, MJI

Revision for important intellectual content: NFRS, HHH, NAHH, SO, NJO, SBSAF, EO, MJI

Supervision: NAHH, HHH, MKAK, MJI

All authors read and approved the final manuscript.

None.

Raw Ct files and individual tumour-volume datasets are unavailable due to historical data-storage limitations. However, all processed data used for analyses are fully presented in the manuscript and supplementary materials, and additional information can be provided upon reasonable request.

All procedures were performed in accordance with Research Animal Ethic Committee UiTM (UITM CARE: 316/2020) guidelines.

Not applicable.

Written informed consent was obtained from the patient’s mother for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

The authors declare that generative AI tools were used solely to improve the clarity and language of selected sentences in the manuscript. The use of AI was limited to linguistic refinement and did not involve the generation of scientific content or data analysis. The authors take full responsibility for the accuracy, originality, and integrity of the work.

The authors declare that they have no competing interests.