Iodine-125 brachytherapy triggers immunogenic cell death and potentiates anti-PD-L1 immunotherapy in bone metastatic triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) with bone metastasis is challenging to treat due to an immunosuppressive microenvironment and the limited, transient immune activation from conventional external beam radiotherapy. Inducing immunogenic cell death (ICD) offers a strategy to remodel this environment, but its systemic effects remain to be validated. Here, we investigate iodine-125 ((125)I) brachytherapy as a method to induce sustained ICD and activate systemic antitumor immunity in TNBC bone metastasis using transcriptomic analysis, a dual-tumor model, and CALR-targeted molecular imaging. METHODS: A dual-tumor model of 4T1 mouse breast cancer with tibial bone metastasis and distal subcutaneous implantation was established. The therapeutic efficacy of (¹²⁵)I seeds at different doses (0.3-0.8 mCi), alone or in combination with anti-PD-L1 antibody, was systematically assessed. ICD biomarkers (CALR, HMGB1) and immune cell infiltration were analyzed using Western blot, qRT-PCR, immunohistochemistry, and flow cytometry. Transcriptome sequencing was performed to explore changes in ICD and related immune pathways. In vivo dynamic imaging of ICD was achieved using a CALR-targeting bimodal nanoprobe. Comparative analyses were conducted between (125)I brachytherapy and EBRT to assess differences in tumor control and immune activation. Treatment sequencing studies were performed to evaluate the optimal combination strategy. RESULTS: This study confirmed that (125)I seeds effectively induce sustained immunogenic cell death, significantly upregulating damage-associated molecular patterns (DAMPs) such as CALR and HMGB1. In the 4T1 bone metastasis model, local implantation of (125)I (0.3-0.8 mCi) inhibited tumor growth in a dose-dependent manner, with the 0.8 mCi group showing the best therapeutic effect and no significant toxicity. Mechanistically, (125)I promoted CD8α⁺T cell infiltration, activated ICD-related pathways, and triggered systemic antitumor immunity, as demonstrated by inhibition of distant tumor growth in the bilateral tumor system. Compared with EBRT, 125I brachytherapy achieved superior local control and more robust immune activation. Using a CALR-targeting nanoprobe, dynamic in vivo imaging of ICD was successfully achieved. Treatment sequencing studies revealed that initiating with (125)I brachytherapy followed by combination therapy with PD-L1 antibody and Abraxane resulted in the most favorable outcome, confirming this "(125)I-first" approach as an effective immune priming strategy. DISCUSSION: This study demonstrates that (125)I brachytherapy overcomes the limitations of conventional radiotherapy by inducing sustained immunogenic cell death, remodeling the tumor immune microenvironment, and activating systemic antitumor immunity. (125)I seeds not only inhibit the growth of TNBC bone metastases and distal tumors but also significantly enhance the infiltration of CD8α(⁺)T cells into tumor sites. Notably, the sequential regimen of (125)I brachytherapy followed by PD-L1 antibody and Abraxane shows significant synergistic effects. These findings establish (125)I brachytherapy as a superior immune-priming modality and provide a mechanistic rationale for integrating low-dose-rate brachytherapy with immunotherapy to overcome resistance in bone-metastatic TNBC, offering strong potential for clinical translation., iodine-125 seeds, triple-negative breast cancer.