C57BL/6J mice (female, 6 weeks old,20g) were obtained from Chongqing Tengxin Biotechnology Co., Ltd. (Chongqing, China). In immunological experiments, female mice are preferred due to the significant impact of androgens on immune indicators. All animal experiments were approved by the Animal Care and Ethics Committee of Southwest Medical University (Luzhou). Lewis lung carcinoma (LLC) cell line was purchased from (Cell Bank of the Chinese Academy of Sciences, China) and the cells were cultured in Dulbecco’s modified Eagle medium (L110KJ, BasalMedia) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cell cultures were incubated in a humidified incubator at 37 °C and 5% CO₂.

A tumor model was developed by injecting 100ul of 1×10^⁶.LLC cells subcutaneously into a mouse’s right leg. Radiotherapy was administered once the tumor reached 250 mm³, with volume calculated using the formula: length × width × width × 0.5. After ear-tagging the mice, randomization was done using Excel’s random number generator. Each group had 5 mice to adhere to the “reduction” principle of the 3Rs, using the minimum number needed for clear results. The groups were labeled as control, H-BT, H-RT, and L-TBI+H-BT. To minimize bias in quantitative assessments, we ensured objective measurements like animal weight and biochemical indicators were directly generated by instruments, reducing human error. For subjective assessments, particularly histopathology scoring, we enforced blinding by having tissue sections randomly coded by another researcher and evaluated by a pathologist unaware of group assignments.

The mice were fixed in our home-made small animal radiotherapy box, exposing the whole right leg through a small hole, leaving the right leg in a stretched state and the left leg in a natural state. All right legs and primary tumors were placed in the radiation field for radiotherapy. Our radiotherapy box had been tested in an ionization chamber before radiotherapy. Dose rates in the center and middle planes of the irradiation field were measured. In addition, thermoluminescence films were stacked near the tumor to verify the dose. L-TBI or H-RT was delivered with a 6MV linear accelerator (VarianClinac600C, USA) at a source-to-surface distance of 100 cm. L-TBI was whole-body irradiation at a dose rate of 24cGy/min, a dose rate of 0.1 Gy/min, and a primary H-RT of 8Gy×3, a dose rate of 400cGy/min.

Mice were anesthetized with 2% isoflurane for induction and 1% isoflurane for maintenance using a small animal anesthesia machine (Reddot R540, China). The needle was inserted into the tumor’s distal edge after taping the mouse’s limbs. The needle was cleaned with an iodophor wipe between applications. The 192Ir high dose rate source was dispensed by a remote afterloader (Shinav, XHDR39) into the needle placed at the tail of the tumor. Brachytherapy prescription was 8 Gy proximal to or cranial to the margin of the tumor. (Depth range 5-10mm, depending on tumor size; dose rate ≥0.12Gy/min, depending on age of 192Ir source). Medical physics staff conducted dose calibration and quality assurance checks on the irradiators as follows:

The radioactive source used in this study is iridium-192 (¹⁹²Ir). The precise half-life of this source is 73.83 days (commonly approximated as 74 days in clinical practice). The detector used to measure the source’s output dose rate and perform equipment radioactivity calibration is a well-type ionization chamber, specifically the HDR 1000 PLUS WELL CHAMBER model. This is the dedicated standard equipment for calibrating high-dose-rate brachytherapy sources. we employ the nationally benchmark-calibrated HDR 1000 PLUS WELL CHAMBER well-type ionization chamber to perform periodic absolute measurements and calibrations of the output dose rate for brachytherapy unit radiation sources, ensuring the accuracy of dose delivery.

All daily quality control, periodic calibration, and radiation physics assurance for radiotherapy equipment (including brachytherapy units and planning systems) are strictly executed and documented by our Medical Physics Department in accordance with relevant national regulations and industry standards. This ensures the safety and precision of clinical treatments.

Supplementary Key Parameters for This Study:

Treatment Equipment: Shandong Xinhua Brachytherapy System, Model XHDR30.

Initial Radioactive Source Activity: 10 Ci.

Treatment Planning System: Shandong Xinhua TPS3d10, employing inverse optimization algorithms for plan design.

Dose Targets: Plans are optimized to ensure 80% of the prescribed dose covers the target volume (tumor mass).

Mice were euthanased by cervical dislocation. Tumor and spleen tissues were taken and placed in DMEM medium, and the tissues were cut and ground respectively with scissors at 4° ice. Tumors were then incubated for 20 minutes at 37 °C in digestive enzymes containing 0.2% collagenase IV, 0.01% hyaluronidase and 0.002% DNase I and spleen was lysed in red blood cell lysate for 15 minutes (Biosharp). The cells were then filtered with filter loops, the single cell suspension thus obtained was used to identify viable cells with FVS780-APC-cy7(BD Pharmingen), the entire immune cell population was labeled with CD45-Pe-cy7(BD Pharmingen), and the remaining surface stained was labeled with the following antibodies: CD3-APC, CD8-PE, CD4-FITC, NK1.1-PE, CD11b-APC, CD11c-PE, CD86-FITC, IA-IE-Percp-Cy5.5, CD11b-FITC, GR-1-APC, F4/80-Percp-Cy5.5 antibodies(BD Pharmingen), according to manufacturer’s protocol After surface labeling with all antibodies, cells were treated with a fixation and permeabilization kit (BD Pharmingen) followed by CD206-PE (Biolegend)staining, then washed twice, and finally stained samples were analyzed using Beckman Coulter Gallios flow cytometry (Beckman Coulter, Miami, Florida, USA).

Tumor tissues were fixed in 10% neutral buffered formalin and embedded in paraffin, and sections 4μm thick were cut and used for immunohistochemistry (IHC). Sections were labeled with the following antibodies according to the manufacturer’s instructions: Ki67,γ-H2AX, calreticulin (CALR), (high–mobility group box 1)HMGB1, (.Heat shock protein 70)HSP70. Image-j were taken using a digital slide scanner.

Following deep isoflurane anesthesia, blood was drawn from the mice’s orbital cavity and kept at room temperature for 45 minutes before the mice were euthanized. The blood samples were then centrifuged at 2500 rpm for 20 min to obtain the upper serum layer INF-γ, CXCL10, IL-2, IL-10 levels were measured by a specific antibody ELISA kit according to the manufacturer’s instructions (Abin) by standard ELISA methods.

Statistical analysis was performed using GraphPad Prism V.9.3.1 software. Data are expressed as mean ± SEM. After verifying that the data follows a normal distribution. One-way ANOVA and Tukey’s test compared column means across multiple data sets, while a T-test was used for two data sets, with p-values under 0.05 deemed significant.

Radiotherapy was administered to mice using two protocols: 8 Gy x 3 H-BT and H-RT (Figure 1A). Hematologic toxicity was evaluated by measuring lymphocyte counts. Lymphocyte counts were significantly lower in the H-RT group compared to the control group whereas in the H-BT group, lymphocyte counts were higher than in the H-RT group (Figure 1B). Radiotherapy led to a more pronounced suppression of tumor growth in the H-BT group compared to the H-RT group, accompanied by a concomitant increase in γ-H2AX-positive cells (Figures 1C–E).

We assessed whether H-BT and H-RT induce ICD differently (Figure 2A). Immunohistochemical analysis showed that H-BT significantly increased CALR and HSP70 expression compared to H-RT, while HMGB1 levels rose without significant difference (Figures 2B–D).

The infiltration of immune cells into the tumor site was evaluated. A significantly higher percentage of CD8⁺ T cells was observed in the H-BT group compared to the H-RT group (Figures 3A, B). Although the H-BT group exhibited a greater number of cDC1 cells, this difference did not reach statistical significance (Figures 3C, D). Additionally, the proportion of NK cells in the tumor microenvironment was significantly increased in the H-BT group compared to the H-RT group (Figures 3E, F).

The presence of myeloid-derived suppressor cells (MDSCs) at tumor sites following radiotherapy was assessed. The proportion of MDSCs was significantly lower in the H-BT group than in the H-RT group (Figures 4A, B). In contrast, the H-RT group exhibited a higher proportion of MDSCs compared to the Control group. Furthermore, analysis of macrophage polarization revealed that the H-BT regimen significantly promoted M1 anti-tumor macrophage polarization (Figures 4E, F). Neither H-RT nor H-BT had a significant effect on neutrophil levels (Figures 4C, D).

Develop a bilateral tumor model to assess if low-dose total body irradiation (L-TBI) temporarily boosts systemic immune responses. H-BT was delivered to the tumor on the irradiated side three days after L-TBI (Figure 5A).

In the spleen, L-TBI+H-BT significantly increased the proportion of CD8⁺ T cells (Figures 5B, C) and promoted dendritic cell (DC) proliferation more effectively than H-BT alone (Figures 5D, E). NK cell activation was also significantly enhanced by L-TBI+H-BT (Figures 5F, G).

No significant differences between H-BT and L-TBI+H-BT were observed in splenic MDSCs, tumor-associated neutrophils, or M2 macrophages (Figures 6A–F).

We measured blood levels of tumor-related cytokines. The L-TBI+H-BT group exhibited significantly higher expression of CXCL10 (Figure 7A) and IFN-γ (Figure 7B) compared to the H-BT group. IL-2 levels were also increased in the L-TBI+H-BT group (Figure 7C), whereas IL-10 production was significantly reduced (Figure 7D).

We evaluated if adding L-TBI enhanced the temporary systemic immune response by observing the growth of contralateral non-irradiated tumors. The L-TBI+H-BT combination significantly inhibited tumor growth on the untreated side (Figure 7E), an effect not observed with H-BT alone.