Boron neutron capture therapy preserves immune cells and induces robust anti-tumour immunity in preclinical mouse model

Domain: chemistry
Published: 2026-01-08
Editorial outcome: minor_revision

Abstract
Radiotherapy can both activate and suppress immunity, making it difficult to predict or modulate these opposing effects for better cancer treatment. Boron neutron capture therapy (BNCT), a cellular-level radiotherapy, has demonstrated remarkable therapeutic efficacy in clinical practice, but mechanistically remains inadequately explored. Here, we compare the effects of BNCT with X-ray irradiation at equivalent radiation doses on immune cells and define the immunological mechanisms behind the improved therapeutic benefit of BNCT in mouse tumour models. We find that BNCT has a minimal effect on immune cell viability, while it triggers an immunogenic tumour cell death, ultimately inducing stronger anti-tumour immunity. Additionally, single-cell RNA sequencing indicates that BNCT reshapes the tumour microenvironment by enhancing dendritic cells, T cells, and NK cells activity. Thus, these findings provide important insights into radiobiological mechanisms following BNCT and inform strategies to preserve immune cells during radiotherapy and to increase cancer treatment efficacy.

Introduction
The remodelling of the tumour immune microenvironment induced by radiotherapy is currently garnering extraordinary attention1–3, as it can enhance anti-tumour immune responses but, in some cases, also exerts immunosuppressive effects4. The response status of various immune cells plays a pivotal role in shaping treatment outcomes, yet these cells are inherently highly radiosensitive5. Over the past few decades, despite remarkable advancements in the precision of radiation delivery, whether using X-rays, proton beams, or heavy ions, collateral damage to immune cells within tumour tissues has remained an unavoidable challenge. Low-dose radiotherapy modulates immune cell infiltration but compromises tumour control6. Conversely, high-dose radiotherapy achieves more effective tumour ablation but at the expense of rapid immune cell depletion7. Although high-dose regimens can induce immunogenic cell death, the concurrent loss of immune cells often constrains the potential to activate antigen-presenting cells and tumour-specific T cells6. Thus, the ability to eradicate tumour cells while sparing immune cells represents an attractive but elusive goal, which is a dilemma to balance.

Boron neutron capture therapy (BNCT) is theoretically regarded as a binary-targeted radiotherapy with the potential for single-cell precision killing8–10. When boron-10 accumulates in tumour cell and captures low-energy (~0.025 eV) thermal neutron, it undergoes nuclear capture, resulting in a 10B(n,α)7Li lethal reaction that produces high linear energy transfer (LET) α particle (150 keV/µm) and lithium-7 nucleus (175 keV/µm) with track lengths of only 5–9 μm. Based on this physical principle, BNCT has been hypothesised to selectively destroy tumour cells while sparing neighbouring normal and immune cells in the tumour microenvironment. The combination of high-LET effectiveness and minimal bystander range gives BNCT the distinctive feature of “tumour cell precision blasting”, which may provide a unique radiotherapeutic strategy that simultaneously achieves tumour ablation and immune cell preservation, contributing to the excellent therapeutic effects.

Encouragingly, BNCT has shown remarkable efficacy in clinical trials, particularly in patients with inoperable malignant tumours11, such as head and neck cancer12,13, glioblastoma multiforme14–17, and malignant melanoma18–21. In 2020, BNCT received clinical approval to treat locally advanced or recurrent unresectable head and neck cancer, marking a significant milestone. In real-world clinical practice, the objective response rate has reached 80.5%, and the 1-year overall survival rate is 75.4%22, substantially outperforming traditional treatment options23,24. And a single treatment session is typically sufficient, offering convenience to patients. These impressive outcomes raise important questions regarding the biological basis for the therapeutic advantage of BNCT. However, despite the promising clinical outcomes, the underlying radiobiological mechanisms of BNCT remain insufficiently explored. The concept of BNCT as a single-cell radiotherapy strategy has largely remained theoretical, with limited direct biological evidence to support immune cell sparing in vivo. Moreover, although preliminary reports suggested the possibility of abscopal effects with BNCT25, they failed to explain the immunological mechanisms. Neither the occurrence of BNCT-induced immune activation nor its underlying mechanisms has been systematically investigated, limiting its broader integration into mainstream clinical practice.

In this study, we take a step forward in addressing this gap by providing direct experimental evidence for the single-cell radiotherapy concept and by elucidating the immune mechanisms engaged by BNCT. We demonstrate that BNCT achieves tumour cell-selective killing while avoiding damage to immune cells and triggers immunogenic tumour cell death, thereby enabling robust activation of systemic anti-tumour immunity with long-term memory. Single-cell transcriptomic profiling of BNCT-treated hot and cold tumours reveals enhanced antigen processing and presentation by dendritic cells, increased CD8+ T-cell activity, and augmented NK-cell cytotoxicity, and we identify CD8+ T cells as essential mediators of BNCT-induced tumour eradication. Together, these findings establish the immunological significance of BNCT and provide a rationale for preserving immune cells as a mechanistic driver of improved therapeutic benefit.

Results
BNCT triggers robust systemic anti-tumour immunity and shapes immunological memory
BNCT is a type of cellular-level radiotherapy (Fig. 1a,b). Malignant melanoma is a clinical indication for BNCT. In this study, we used B16F10 cells, a highly metastatic murine melanoma cell line. B16F10 tumour models were constructed, which are commonly used as a classic model of immunologically cold tumours in tumour immunology26, characterized by an immunosuppressive profile. In addition, we have established an immunologically hot tumour model using the MC38 murine colorectal cancer cell line. With its relatively shallow location, colorectal cancer also shows potential as a suitable candidate for BNCT.

The boron carrier agents for BNCT need to meet the following requirements: high tumour uptake (>20 ppm), high tumour selectivity (tumour to normal tissue ratio > 3), and low toxicity27–29. [10B]BPA (boronophenylalanine)30,31, an approved drug, is the most widely used boron carrier agent in clinical practice. At the cellular level, BPA resulted in high tumour cell uptake (Supplementary Fig. 1a). In vivo, boron uptake in the B16F10 and MC38 tumour tissues was 7.74 ppm and 12.60 ppm, respectively, one hour after intravenous injection of 100 mg/kg [10B]BPA (Supplementary Fig. 1b,c).

First, we tested the efficacy of BNCT in these two models. The average therapeutic doses were 4.395 Gy for the B16F10 tumour model and 4.029 Gy for the MC38 tumour model, respectively (Supplementary Fig. 1d). Following anaesthesia, the mice were positioned in a specialized mold, with the tumours near the neutron beam, for 30 min of neutron irradiation (Supplementary Fig. 1e). X-ray (4 Gy) external irradiation was used as a control group, BNCT significantly inhibited the growth of B16F10 tumours (Supplementary Fig. 1g,h), and completely cured MC38 tumours (Supplementary Fig. 1i,j), indicating that BNCT is highly effective in treating both tumours.

Given the theoretical basis for the ability of BNCT to preserve immune cells, we next investigated the anti-tumour immune effect of BNCT. Abscopal effects were assessed, and tumour cell vaccines and rechallenge models were established to explore the anti-tumour immunity induced by BNCT. These models have also been used to assess immunogenic cell death (ICD) in immunocompetent syngeneic systems in vivo32,33. Localized radiotherapy may lead to distant, non-irradiated tumour regression, known as the abscopal effect, which has been connected to immune system-mediated mechanisms34,35. B16F10 or MC38 tumours were implanted in the right shoulder of each mouse, and half of the tumour cells from the same source were implanted in the left lower abdomen. Seven or ten days later, BNCT treatment was applied, with only the shoulder tumour exposed to the thermal neutron beam (Fig. 1c,f). In both models, BNCT inhibited the growth of the distal tumour, not just the primary tumour, whereas 4 Gy localized X-ray treatment did not affect the distal tumour (Fig. 1d,e,g,h). Notably, even with the same BPA injection and neutron irradiation on the right shoulder, the growth of the distal tumour was not suppressed without the primary tumour (Fig. 1h). These findings suggested that the BNCT-treated tumour induced secondary effects that inhibited the distal tumour.

Whether BNCT-treated tumour cells could function as tumour vaccines was further investigated. The cells were incubated overnight with 10 mM BPA, irradiated with thermal neutrons for 10 min (Supplementary Fig. 1f), and injected into the mice. Ten days later, untreated tumour cells were implanted in the contralateral site (Fig. 1i). Preinjection of BNCT-treated B16F10 cells delayed the growth of secondary tumours (Fig. 1j) and prolonged the survival of tumour-bearing mice (Fig. 1k). BNCT-treated MC38 cells completely suppressed the growth of the second tumour (Fig. 1l) and allowed the mice to remain healthy (Fig. 1m). These results demonstrated that BNCT could convert irradiated tumour cells into an immunogenic hub and help tumour cells become more visible to the immune system.

Rechallenge experiments were performed to further investigate whether BNCT could promote long-term immune memory (Fig. 1n). MC38 tumour cells were inoculated on the right side of the mouse and treated with BNCT. After 10 weeks, the mice were subjected to a second tumour challenge on the left side. As shown in Fig. 1o, BNCT treatment completely suppressed secondary tumours, and the mice survived longer (Fig. 1p). Even in the rechallenge experiment conducted at week 20, BNCT treatment still completely inhibited tumour growth (Supplementary Fig. 1k,l). Moreover, in the treatment group at 10 weeks post-treatment, the percentage of naïve CD8+ T cells decreased, the percentage of central memory T cells (Tcm) among CD8+ T cells in the blood increased from 19% to 26%, and the percentage of effector memory T cells (Tem) significantly increased from approximately 7% to 12% when compared with those in the control group (Fig. 1q, Supplementary Figs. 1m,2). A similar trend was also observed in the axillary lymph nodes and spleen (Supplementary Fig. 1n–q). These findings suggested that BNCT could prime systemic anti-tumour immunity with durability and had the potential immunological benefits by inducing ICD to reduce the systemic disease burden.

BNCT evokes enhanced immunogenic cell death
To gain insight into the immunogenic cell death induced by BNCT, we characterized the properties of BNCT-treated tumour cells and molecular markers. First, we investigated the morphological changes following BNCT treatment, shown in Fig. 2a and Supplementary Fig. 3a,b. The cells swelled 96 h after BNCT treatment in vitro, a phenomenon that was not prominent within the initial 48 h (Supplementary Fig. 3c). In addition, BNCT-treated MC38 cells displayed fragmentation, with abundant cell debris observed in the culture medium. By contrast, cells exposed to X-ray irradiation demonstrated only minimal morphological alterations. Cell viability assays via CCK-8 revealed significant cytotoxic effects of BNCT on both cell types (Fig. 2b,c). Notably, this cytotoxicity was not immediate; a reduction in cell viability to approximately 50% required 72–96 h. The morphological changes in tumour tissues treated with BNCT in vivo were consistent with those observed in vitro. Five days post-treatment, the tumour tissues exhibited a loose structure with reduced cellular integrity (Fig. 2d). Certain regions displayed more severe cell damage, characterized by a “reticular” structure in cellular arrangements (Fig. 2e). Compared with the NC and X-ray groups, the BNCT-treated tumours presented significantly larger average cell sizes (Fig. 2f), progressively increasing intercellular spaces (Fig. 2g), reduced cell density (Supplementary Fig. 3d), and enlarged nuclear areas (Supplementary Fig. 3e). The changes in intercellular spaces and cell density became more pronounced over time, reflecting the progressive disruption of tumour architecture by BNCT. In addition, transmission electron microscopy revealed clear evidence of structural disruption of mitochondria in BNCT-treated cells (Supplementary Fig. 3a,b). JC-1 staining revealed that the aggregate to monomer ratio decreased in BNCT-treated B16F10 cells and MC38 cells compared with controls (Supplementary Fig. 3f–h), indicating a loss of mitochondrial membrane potential. This decrease was consistent with the morphological changes observed by electron microscopy, suggesting mitochondrial dysfunction induced by BNCT.

BNCT has been demonstrated to elicit a systemic anti-tumour immune response, suggesting that the death of tumour cells is an immunogenic form of cell death. We scrutinized ICD markers at the cellular and molecular levels to substantiate this, focusing on damage-associated molecular patterns (DAMPs)32,33,36. Extracellular ATP acts as a “find-me” signal that recruits DCs and macrophages, promoting proinflammatory responses33. Following BNCT treatment, the extracellular ATP levels in B16F10 cells (Fig. 2h) and MC38 cells (Fig. 2i) gradually increased relative to those in the control group, peaking at ~84 h and 60 h, respectively. At the peak, ATP levels in the culture medium of the BNCT group were approximately four times greater than those in the untreated control group and two times greater than those in the X-ray group, demonstrating that BNCT triggered ATP release, a conserved observation across both B16F10 and MC38 cells. High-mobility group box 1 protein (HMGB1), which can mediate potent immunostimulatory effects33, was also evaluated. Compared with those in the NC group, the HMGB1 expression and cytoplasmic accumulation in tumour cells were significantly greater in vivo at days 1, 3, and 5 post-BNCT (Fig. 2j,k), whereas X-ray treatment only slightly elevated HMGB1 levels (Fig. 2j,k). In contrast, calreticulin exposure was not significantly induced after BNCT (Supplementary Fig. 4a,b). We subsequently evaluated interferons (IFN) in mouse serum, which were critical immunostimulatory molecules. In B16F10 tumour-bearing mice, there was a trend toward increased expression of IFN-α/β/γ after BNCT treatment on day 7 (Fig. 2l). Similarly, in MC38 tumour-bearing mice, BNCT significantly elevated the serum levels of IFN-α/β/γ on day 5, with IFN-α levels increasing by more than 10-fold (Fig. 2m). The upregulation of ATP release, HMGB1 expression, and interferon level collectively further confirmed that BNCT induces immunogenic cell death in tumour cells.

DNA double-strand breaks (DSBs) are recognised as the critical mechanism underlying the cytotoxic effects of high LET particles, including α particles. We observed a significant increase in DSBs in the BNCT group compared with those in the NC group (Supplementary Fig. 4c,d). At 1-, 3-, and 5 days post-treatment, the fold increases in DSBs for the BNCT group were 149, 211, and 205, respectively, whereas for the X-ray group, the corresponding values were 90, 30, and 2, respectively. These findings indicated that the DNA DSBs induced by BNCT were more severe and persistent, remaining irreparable for at least 5 days, distinguishing them from the transient effects observed with X-ray therapy. This finding was further supported by western blot analysis (Supplementary Fig. 4e). In vitro experiments revealed that BNCT induced cell cycle arrest, which began 12 h after treatment and persisted for up to 96 h (Supplementary Fig. 4f–h). We further explored the mechanisms underlying BNCT-induced cell death (Supplementary Fig. 4i,j) compared to X-ray radiation (Supplementary Fig. 4k,l). The inhibition of caspase proteins failed to rescue the cell death induced by BNCT, whereas pan-caspase inhibitors were able to rescue some cells from death under X-ray irradiation. Similarly, inhibitors of necroptosis and ferroptosis could not prevent cell death caused by BNCT. These results indicated that BNCT-induced cell death was not associated with apoptosis, necroptosis, or ferroptosis. Given the characteristics of BNCT-induced cell death, such as cellular swelling, increased intercellular spacing, reduced cell density, persistent and irreparable DNA double-strand breaks, and prolonged cell cycle arrest, we hypothesized that the mode of cell death triggered by BNCT might be necrosis.

BNCT preserves and recruits immune cells within the tumour microenvironment
BPA is an analogue of phenylalanine, which is taken up mainly through L-type amino acid transporter 1 (LAT1), which is encoded by the Slc7a5 gene and is overexpressed in many tumour cells37–40. Thus, boron selectively accumulates in tumour cells but remains scarce in immune cells. When exposed to the neutrons, tumour cells are far more likely to undergo boron neutron capture reactions than immune cells are, enabling precise tumour-cell killing while sparing immune cells (Fig. 3a).

To clarify the selective accumulation of boron in tumour cells, we examined the expression of Slc7a5 in non-immune (CD45-) and immune (CD45+) cells in the B16F10 and MC38 tumour models. We found that the expression level of the Slc7a5 gene in non-immune cells was 10.07 times (Fig. 3b) and 10.41 times (Fig. 3c) higher than in immune cells, respectively, indicating the selective enrichment of boron in non-immune cells, greatly reducing the probability of a boron neutron capture reaction in immune cells. Additionally, the uneven distribution of BPA within the tumours in vivo was confirmed via MALDI-TOF imaging (Fig. 3d). In regions with blood flow where immune cells were concentrated, the BPA accumulation was low.

Assessing cell viability within the tumour provides direct insight into the preservation of immune cells by BNCT. Two days after BNCT treatment, the viability of non-immune cells (CD45-) slightly declined (Fig. 3e), and by day 5, their activity had decreased to below 40% (Fig. 3f), confirming the tumour-killing efficacy of BNCT. In contrast, the viability of immune cells (CD45+) remained unchanged compared with that of the control group at both time points (Fig. 3e,f), underscoring the ability of BNCT to effectively eliminate tumour cells without harming immune cells within the tumour microenvironment. On the other hand, X-ray treatment was less effective than BNCT in eradicating tumour cells, causing substantial damage to immune cells (Fig. 3e,f), with this damage persisting for at least 5 days, highlighting BNCT’s unique advantage of selective tumour-cell killing while preserving immune cell function.

Furthermore, immunofluorescence analysis (Fig. 3g–n) revealed increased numbers of infiltrating immune cells in the tumour tissue following BNCT treatment. Compared with untreated tumours, BNCT-treated tumours exhibited increased infiltration of three types of immune cells: CD8+ T cells, CD11c+ (particularly CD11c+MHC-II+) myeloid cells, and F4/80+ (especially F4/80+MHC-II+) macrophages. Compared with X-ray treatment, tumours under BNCT increased infiltration of CD8+ T cells, CD11c+MHC-II+ myeloid cells, and F4/80+MHC-II+ macrophages. These findings suggested that immune cells were not directly affected by BNCT and that indirect mechanisms might be involved in their recruitment.

scRNA-seq reveals inflammatory cancer cell subclusters following BNCT
Since BNCT is a single-cell killing therapeutic modality, we employed single-cell RNA sequencing (scRNA-seq) to analyze the tumour microenvironment after BNCT treatment. For the immunologically cold B16F10 tumour, all tumour cells were harvested one day after BNCT treatment. On day 7, CD45+ immune cells were enriched at a ratio of CD45- to CD45+ was 1:2, followed by library preparation, sequencing, and analysis (Fig. 4a). After data filtering, 63,415 cells were captured in the B16F10 tumour experiment: 10,679 from the NC (negative control) group, 8478 from the X-ray group, and 14,278 from the BNCT group on day 1; 14,356 from the NC group, 8758 from the X-ray group, and 6902 from the BNCT group on day 7. Clustering was performed, and cell populations, including cancer cells, DCs, T cells, NK cells, B cells, fibroblasts, endothelial cells, macrophages, and monocytes, were annotated on the basis of marker gene expression (Fig. 4b, Supplementary Figs. 5a–c, 6a).

Differentially expressed gene (DEG) analysis of tumour cells following BNCT treatment (Supplementary Fig. 6b,c) revealed that compared with those in the X-ray group, tumour cells on day 1 presented upregulated expression of cell cycle-related genes, such as Ccng1 and Cdkn1a. Gene Ontology (GO) analysis of enriched pathways revealed an increase in the G1 DNA damage signalling pathway and DNA damage checkpoint signalling in the BNCT group (Fig. 4c). Analysis of the cell cycle (Supplementary Fig. 6d) also indicated that relative to the NC group, BNCT treatment increased the proportion of tumour cells in the G1 and G2/M phases, with a greater percentage of G2/M phase cells than in the X-ray group. Notably, the increase in G1 phase cells persisted until day 7 in the BNCT group. In contrast, the cell cycle of X-ray-treated cells was similar to that of the control group, suggesting that BNCT caused more severe and persistent cell cycle damage, impairing tumour cell proliferation. More importantly, on day 7 post-BNCT, tumour cells presented significant upregulation of immune-related genes, such as the Tnf, Irf8, Ifitm3, and Irgm1 genes, which were enriched in response to interferon pathways (Fig. 4c, Supplementary Fig. 6c).

Owing to the heterogeneity of the boron distribution and the spatial complexity of the tumour tissues, we further clustered the tumour cells (Fig. 4d, Supplementary Fig. 5d, 6e). Compared with those in the X-ray treatment group, the highly proliferative c0_Mki67 subcluster, characterized by high expression of cell cycle-related genes, such as Mki67, Top2a, and Cdk1 (Supplementary Fig. 6g), decreased from 40% to 25% on day 1 post-BNCT. By day 7, the proportion of the c0_Mki67 subcluster in the X-ray group was similar to that in the NC group. In contrast, this subcluster further declined to below 10% in the BNCT group (Supplementary Fig. 6f), suggesting that BNCT treatment more effectively disrupted the activity of highly proliferative cells, with a stronger and more sustained impact than X-ray treatment. The c1_Fgfr2+ subcluster had no obvious changes in proportion (Supplementary Fig. 6h). The c2_Jund+ subcluster, which was characterized by its high expression of the Jund gene, a known stress-related regulator of cell cycle progression41, displayed G1 phase cell cycle arrest (Supplementary Fig. 6i,j), and increased from 20% to 30% on day 1 following both BNCT and X-ray treatments. By day 7, the proportion of these cells continued to increase in the BNCT group, whereas in the X-ray group, it decreased to levels comparable to those in the NC group, which was consistent with the observed changes in the cell cycle distribution (Supplementary Fig. 6d).

The c3_Rp+ subcluster was characterized by a lower number of expressed genes and upregulation of ribosomal-related genes, such as Rps29 and Rps27. The c3_Rp+ subcluster accounted for more than 30% of the cells in the BNCT group on day 7, compared with only approximately 10% in the X-ray and NC groups (Fig. 4e), and exhibited immune-related signatures (Supplementary Fig. 6k). The total expressed gene number of cells may reflect the extent of cellular damage42. Different clusters presented various numbers of expressed genes (Fig. 4f). The c0_Mki67+ subcluster, representing highly proliferative cells, exhibited the greatest number of expressed genes. While compared with the c0_Mki67+ subcluster, the c1_Fgfr2+ (Supplementary Fig. 6e) and c2_Jund+ subclusters approximately presented two-thirds of the total expressed counts within the c0_Mki67+ subcluster, possibly suggesting the mild damage induced by BNCT. Notably, the c3_Rp+ subcluster showed a markedly reduced expressed gene number, which may indicate severe damage. Further analysis of the c3_Rp+ subcluster’s gene expression profile also revealed the pathway enrichment of the inflammatory response and granulocyte chemotaxis (Fig. 4g). Additionally, this subcluster showed markedly greater expression of certain cytokines, including the Ccl8 and Ccl5 genes, than the other subclusters did (Supplementary Fig. 6l).

We also analyzed the immunologically hot tumour model MC38 via scRNA-seq. Tumour cells were collected 5 days after BNCT or X-ray treatment (Fig. 4h), capturing 24,910 cells, including 7894 from the NC group, 9580 from the X-ray group, and 7436 from the BNCT group. After clustering the tumour cells (Fig. 4i, Supplementary Figs. 7a–c, 8a), we observed that tumour cells treated with BNCT exhibited an inflammatory phenotype, with the upregulated genes significantly enriched in pathways related to the response to interferon and chemokines (Fig. 4j, Supplementary Fig. 8b), compared with those in the X-ray group. Additionally, we identified a subpopulation denoted as c3, which increased in proportion following BNCT treatment (Fig. 4k). This cluster had a reduced number of expressed genes (Fig. 4l) and was enriched in the neutrophil and granulocyte chemotaxis pathways (Fig. 4m). Notably, the two c3 subclusters from B16F10 and MC38 tumours shared 40 overlapping genes among the top 100 specifically upregulated in the BNCT group compared with the X-ray group (Fig. 4n, Supplementary Fig. 8c), including some genes encoding cytokines such as Ccl2, Ccl8, Ccl12, Cxcl2, Ccl9, and Ccl6. In summary, single-cell RNA sequencing revealed a unique subpopulation of tumour cells exhibiting a low expressed gene number, with an increased proportion following BNCT treatment. These cells demonstrated significant upregulation of immune-related genes, specifically cytokines, a phenomenon conserved across the B16F10 and MC38 tumour models. These cells likely serve as critical triggers for the induction of BNCT-mediated anti-tumour immunity.

BNCT remodels the tumour microenvironment and stimulates DCs, T cells and NK cells
The tumour-killing effects of BNCT are intricately linked to the involvement of the immune system. Therefore, we next focused on the dynamics of immune cell populations within the tumour microenvironment. As pivotal antigen-presenting cells, DCs play a critical role in initiating and orchestrating immune responses3. These cells are capable of recognizing and processing antigens and then presenting them to T cells to activate specific adaptive immune responses.

In the immunologically cold B16F10 tumour model (Supplementary Fig. 9a), DCs exhibited significant gene expression changes relative to those in the X-ray group at 7 days post-BNCT treatment, as depicted in Fig. 5a. These genes were enriched in pathways associated with antigen processing and presentation, interferon and interleukin-1 response, and DC chemotaxis (Fig. 5b), as interferon and interleukin-1 are key stimulators of DC maturation. While antigen processing and presentation functions in DCs were not significantly enhanced in the BNCT group on day 1 post-treatment (Supplementary Fig. 9b), there was a marked improvement by day 7 compared with both the NC and X-ray groups (Fig. 5c). Concurrently, genes such as Ccl8, Cxcl2, Ccl2, and Ccl6 were significantly upregulated (Fig. 5d), and they could recruit various immune cells to promote the immune response (including immature DCs, monocytes, and T cells). In the immunologically hot MC38 tumour model (Supplementary Fig. 9c), DCs within BNCT-treated tumour tissues also exhibited upregulated antigen processing and presentation functions (Fig. 5e). These findings suggested that BNCT treatment induced the activation and functional enhancement of DCs within the tumour microenvironment in a time-dependent manner, which might be correlated with the delay in BNCT-induced cell death. We established an in vitro co-culture system to assess DC antigen-presenting capacity (Fig. 5f). Bone marrow derived-dendritic cells (BMDCs) indirectly co-cultured with BNCT-treated B16F10 cells for 3 days showed significantly increased CD80 (Fig. 5g) and CD86 (Fig. 5h) expression compared with control or X-ray groups. When these DCs were subsequently co-cultured with spleen-derived CD8+ T cells, the proportion of CD25+CD69+ T cells increased after 24 h (Fig. 5i), indicating enhanced T-cell activation. Flow cytometry analysis of DCs in tumour tissues revealed increased expression levels of the costimulatory factors CD80 and CD86 after BNCT treatment in both B16F10 (Fig. 5j,k, Supplementary Figs. 9d, 10) and MC38 tumours (Fig. 5l,m, Supplementary Figs. 9e, 11), indicating an enhanced ability of DCs to activate T cells, further supporting the role of BNCT in promoting an immune-active tumour microenvironment.

T cells are central mediators of the adaptive immune system in the fight against cancer. In particular, CD8+ cytotoxic T cells are the most powerful effectors of the anticancer immune response and constitute the backbone of cancer immunotherapy3. We further focused on T-cell changes in the tumour microenvironment after BNCT treatment and clustering analysis of T cells in B16F10 tumour tissues was performed (Supplementary Fig. 9f–h). Although the composition of T-cell subsets did not significantly change (Supplementary Fig. 9i), the CD8+ T cells exhibited an overall activated phenotype following BNCT treatment (Supplementary Fig. 9j). Compared with those in the NC and X-ray groups, the CD8+ T cells in the BNCT-treated MC38 tumours exhibited a significantly increased activation signature (Fig. 6a) and a reduced exhaustion signature (Fig. 6b) 5 days after BNCT treatment. The activation of CD8+ T cells in MC38 tumours was consistent with that in the B16F10 tumour model, demonstrating that the activation effect of BNCT on CD8+ T cells is conserved across tumour models.

T cell activation require extracellular stimulatory signals that are mediated mainly by T-cell receptor (TCR) complexes, which function in tumour antigen recognition and signal transduction by interacting with major histocompatibility complex (MHC) molecules43. Radiation-driven immunogenic cell death may facilitate “antigen spread”44, which could diversify the TCR repertoire. While TCR expansion has been shown to play a crucial role in anti-tumour immunity45, TCR sequencing of T cells from BNCT- or X-ray-treated MC38 tumours was performed to assess the TCR repertoire, which reflecting the dynamics of T cells. Following BNCT treatment, the proportion of expanded T cells (~20%) was greater than that in the X-ray group (<10%), with no expanded T cells detected in the NC group (Fig. 6c). These findings indicated that both BNCT and X-ray treatments can promote the expansion of T cells, with BNCT having a stronger effect. Additionally, analysis of the TCR repertoire composition (Fig. 6d) revealed that X-ray treatment favoured the expansion of the specific T cell clones. In contrast, BNCT treatment increased the expansion and diversity of the TCR repertoire globally. These findings suggested that BNCT may expand tumour antigen pools and facilitate the presentation of a broader range of tumour-specific antigens, enhancing the ability of the immune system to recognize and attack tumour cells.

Flow cytometry analysis of MC38 tumour tissues following BNCT treatment revealed a decreased proportion of CD4+ T cells (Fig. 6e) and an increased proportion of CD8+ T cells (Fig. 6f). The ratio of CD8+ to CD4+ T cells was also elevated (Fig. 6g). Moreover, BNCT treatment increased the infiltration of cytotoxic CD8+ T cells (Fig. 6h), which exhibited a clustered distribution.

Next, we explored the necessity of CD8+ T cells in BNCT-induced anti-tumour immunity. When BNCT was combined with CD8-neutralizing antibodies during the first 8 days post-treatment, the anti-tumour effect was comparable to that of BNCT treatment alone (Fig. 6i,j). However, starting from day 10, the tumours in the group treated with both CD8-neutralizing antibodies and BNCT grew continuously, leading to the death of the mice due to the tumour burden (Fig. 6k). Furthermore, FTY720 can inactivate sphingosine-1-phosphate (S1P) receptors on T cells, inhibiting T-cell egress from lymphoid organs46. The inhibition of T-cell migration by FTY720 resulted in a weakened tumour-killing ability of BNCT, with continuous tumour growth in mice (Fig. 6l,n), indicating that T cells in lymphoid tissues were also necessary for the anti-tumour effects of BNCT. Both experiments demonstrated that within a certain period after BNCT treatment (approximately 10 days), BNCT suppressed tumours through the direct cytotoxic effect of high-LET radiation. Over time, BNCT-induced anti-tumour immunity gradually played the leading role, with CD8+ T cells and T cells in lymphoid tissues essential.

CD8+ T cells interact with MHC class I (MHC-I) molecules on tumour cells, which present antigenic peptide fragments. We observed that both on day 1 and day 7 post-treatment, the overall expression of MHC-I molecules in tumour cells was significantly greater than that in the control group for both the BNCT and X-ray treatment groups (Supplementary Fig. 12a,b), with a more pronounced increase in the BNCT group. Additionally, several genes encoding MHC-I molecules, particularly the key genes H2-K1 (Supplementary Fig. 12d) and H2-D1 (Supplementary Fig. 12e), were significantly upregulated in the BNCT group (Supplementary Fig. 12c). Similar observations were also observed in MC38 tumours, where BNCT treatment led to a significant increase in MHC-I molecule expression (Supplementary Fig. 12f). These findings suggested that BNCT might increase the level of peptide presentation by increasing the surface expression of MHC-I on tumour cells, thereby improving the ability of tumour cells to be recognized by T cells, which contributed to its tumour-killing efficacy.

Natural killer (NK) cells exhibit potent cytotoxicity and secrete cytokines and chemokines such as IFN-γ and TNF, which aid B-cell and T-cell responses and modulate the functions of DCs and neutrophils, thus inducing systemic anti-tumour immunity3. In the B16F10 and MC38 tumour models, NK cell cytotoxicity in the BNCT groups was significantly elevated compared with that in the NC and X-ray groups (Supplementary Fig. 12g,h). Moreover, the expression of cytotoxicity-related genes, such as Granzyme B (Gzmb) and Interleukin-18 (Il-18), was significantly upregulated (Supplementary Fig. 12i,j). These findings indicated that NK cells in the tumour microenvironment were activated following BNCT treatment. This could result in enhanced anti-tumour function, which was conserved across both “cold” and “hot” tumour models. However, the depletion of NK cells did not affect the therapeutic efficacy of BNCT, indicating that although NK cells could be activated after BNCT, they were not essential for the anti-tumour immune effects (Supplementary Fig. 12k–m).

Methods
Mice
All animal experiments were conducted in accordance with the Animal Protection Guidelines of Peking University, China. Animal care and experimental procedures adhered to the animal protocols (IACUC ID: CCME-LiuZB-2) approved by the ethics committee of Peking University. 6–8-week-old female (Considering that female mice exhibit lower aggression when living in groups, which are more suitable for group breeding with reduced management difficulty) C57BL/6J (#213) mice were obtained from Vital River Laboratories (Beijing, China) and housed under specific pathogen-free conditions, maintaining a 12-hour light/dark cycle at 24 ± 2 °C and 50 ± 10% relative humidity, with free access to food and water. The experimental and control animals were co-housed. Animals were euthanized according to IACUC-approved endpoints, including a tumour burden exceeding defined limits (estimated >10% body weight or >20 mm in any dimension), tumours that ulcerated or infected, or tumours that interfered with normal bodily functions, significantly impaired gait, or compromised the animal’s ability to obtain food or water.

General protocols of animal studies
The exact sample size is indicated in the legend. All data were included in the analysis; no data were excluded. All results were independently replicated at least three times with similar results. Mice were randomly assigned to experimental groups (randomization followed two criteria: each mouse had an equal probability of being assigned to any experimental group; and the assignment of one mouse to a group did not affect the assignment of other mice to the same group). Appropriate blinding was used during data collection and analysis.

Cell culture
The B16F10 (#CL-0319) cell lines were purchased from Procell. The MC38 cell lines (#SNL-505) were purchased from SUNNCELL. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, C11995500BT, Gibco) supplemented with 10% FBS (fetal bovine serum, SE100-B, Vistech) and 1% penicillin-streptomycin (15070063, Gibco) at 37°C under 5% CO₂. Upon reaching 80% confluence, the cells were treated with PBS (Phosphate buffered saline, M52270, Meryer) and trypsin (25200114, Gibco) and subcultured into a new culture dish.

Tumour growth and treatment
To verify the efficacy of BNCT for a single tumour model, approximately 1×10⁶ B16F10 cells or 5×10⁵ MC38 cells suspended in 100 μL of PBS were implanted subcutaneously into the mice. Tumour volumes were calculated via the following formula: volume = 1/2 × length × width². Tumour volumes were measured daily or every two days, or twice a week. The therapeutic experiment was conducted when the tumour volume reached 40–100 mm³, which was 7 days for B16F10 tumours and 10 days for MC38 tumours after transplantation. To establish a mouse tumour model of the abscopal effect, 1×10⁶ B16F10 cells or 5×10⁵ MC38 cells were suspended in PBS (100 μL) and injected subcutaneously into the right shoulders of C57BL/6J mice, while half of the tumour cells were injected into the left abdomen. Only the tumours on the right side of the mouse were exposed to thermal neutrons. For the mouse tumour vaccine experiments, B16F10 cells or MC38 cells were incubated overnight with 10 mM [10B]BPA and irradiated with thermal neutrons for 10 min. Then, BNCT-treated B16F10 cells (1×10⁶) or MC38 cells (5×10⁵) were subsequently injected subcutaneously into the right shoulder of each mouse. Ten days later, an equal number of untreated tumour cells were injected subcutaneously into the left abdomen of each mouse. For the mouse tumour rechallenge experiments, 5×10⁵ MC38 cells were subcutaneously inoculated into the left abdomen of mice at 10 or 20 weeks after the initial tumour inoculation. At week 10, memory T cells were analysed from the blood, axillary lymph nodes, and spleen, or tumours were reimplanted on the left side of some mice to monitor tumour growth. For the CD8 depletion experiments, 200 μg of anti-CD8 antibody (BE0061, BioXcell) was injected intraperitoneally every three days. For FTY720 treatment, 10 μg of FTY720 (S5002, Selleck) was administered intraperitoneally one day before the initiation of BNCT treatment, followed by 10 μg every other day for two weeks. For NK cells depletion, 200 μg of Ultra-LEAF Purified anti-mouse NK-1.1 Antibody (108759, Biolegend) was administered intraperitoneally one day before the initiation of BNCT treatment, followed by 200 μg every three days.

Tumour and spleen digestion
Tumour tissues were excised and digested with 1 mg/mL collagenase I (SCR103, Sigma) and 0.5 mg/mL DNase I (18047019, Invitrogen) at 37°C for 30 min. The tumour cell suspension was then passed through a 70 μm cell strainer to remove large pieces of undigested tumour. The spleens from the mice were excised and ground using a 70 μm cell strainer, then transferred to a lymphatic separation solution (7211011, Dakewe). The upper layer was centrifuged for 30 min at 800 g after adding 1 mL Roswell Park Memorial Institute 1640 medium (RPMI 1640, C11875500BT, Gibco), with a slow rise and fall during centrifugation. The right axillary lymph nodes of the mice were harvested for lymph node cells, ground with a syringe plunger through a 70 μm cell strainer, and added to RPMI 1640 medium. At room temperature, lymphocytes were centrifuged at 250 × g for 10 min, with a slow rise and fall during centrifugation.

Preparation of the BPA solution
The NaOH solution was prepared and allowed to cool for future use. The NaOH solution was used to dissolve 200 mg of [10B]BPA (80994598, Dongguan HEC). Then 180 mg of fructose was added to the reaction mixture, which was stirred for 10 min. Then, the pH was then adjusted to 7.5, and the solution was sterilized through a 0.22 μm filter.

Cellular and in vivo boron concentrations
For cellular boron uptake, B16F10 or MC38 cells were plated in 6-well plates at a density of 1×10⁶ cells per well and incubated under standard conditions. The cells were treated with 2–20 mM [10B]BPA and incubated overnight. Then, the cells were washed three times with PBS to remove any residual boron from the culture medium, and then the cells were subsequently digested, centrifuged, and washed again. The cell pellets were lysed with 300 μL HNO₃ (112066, TGREAG) at 65°C for 30 min and then diluted with deionized water. The boron concentration was determined via ICP-MS (Inductively Coupled Plasma Mass Spectrometry, NexlON 350X, PerkinElmer).

For determination of the tumour boron concentration, mice bearing B16F10 or MC38 tumours received intravenous injections of 100 mg/kg [10B]BPA solution. 60 min post-injection, the mice were euthanized. Blood and tumours were collected, weighed, and lysed in the microwave-accelerated reaction system with HNO₃ at 140 W for 10 min, and then diluted with deionized water for analysis. The boron concentration in these samples was assessed via ICP-MS.

BNCT of cells and animals
Cells or animals were irradiated at the In-Hospital Neutron Irradiator 1 (IHNI-1), which is based on a miniature neutron source reactor, with a thermal neutron flux of 1.9×10⁹/cm²/s. The information of neutron spectrum can be found in reference 51. The dose rate of boron-10 at 1 ppm is about 0.0124 cGy/s, the total neutron dose rate is 0.0626 cGy/s, and the γ dose rate is 0.0151 cGy/s. For cell irradiation, MC38 and B16F10 cells cultured in a cell culture bottle were incubated with boron carriers overnight and then irradiated for 10 min using thermal neutrons, ensuring that the adherent culture side was positioned close to the beam outlet. For animal irradiation, each mouse in the BNCT group received an intravenous injection of [10B]BPA (100 mg/kg). Before neutron irradiation, mice anaesthetized with tribromoethanol (MA0478, Meilun Bio) were secured onto a custom-made module, with the tumour-bearing side positioned close to the beam outlet. 60 min after [10B]BPA treatment, the mice were irradiated with thermal neutrons for 20 min (MC38-bearing mice) or 30 min (B16F10-bearing mice).

Dosimetry by SERA
The neutron capture cross sections (σ) vary significantly between nuclides. The thermal neutrons are preferentially captured by 10B nuclei (3837 barn), and can also be captured by H (0.332 barn) and N (1.75 barn) of which tissues are mainly composed of. Therefore, the magnitude of the radiation dose of BNCT is complicated due to the radiation field in BNCT consists of several separate radiation dose components including B-10 dose (D_B, from 10B(n,α)7Li reaction), gamma dose (D_γ, from hydrogen 1H(n,γ)2H in tissue), N-14 dose (D_N, from 14N(n,p)14C in tissue) and hydrogen dose (D_H, from 1H(n,n)p in tissue) with different physical properties.

The Monte Carlo computation-based dose calculation was performed with SERA (Simulation Environment for Radiotherapy Applications; Idaho National Laboratory, Idaho Falls, ID), tumours were simulated as the average soft tissue (adult ICRU-33) according to ICRU report 4652.

Calculation of radiation dose of B16F10 tumour and MC38 tumour
The average boron accumulation (Conc_B) in B16F10 tumour and MC38 tumour was 7.74 ppm and 12.60 ppm, respectively, with thermal neutron irradiation time (T) of 30 min and 20 min, respectively. Tumour tissue was simulated as a sphere with a radius of 0.5 cm. By Monte Carlo computation, the dose rates for four radiation components were:

D_B = 0.011 Gy·min⁻¹ppm⁻¹, D_γ = 0.014 Gy·min⁻¹, D_N = 0.019 Gy·min⁻¹, D_H = 0.026 Gy·min⁻¹

The total absorbed dose:
D_total = (D_B × T × Conc_B + D_γ + D_N + D_H) × T

The therapeutic doses were 4.395 Gy for B16F10 tumour model and 4.029 Gy for MC38 tumour model, respectively.

Boron distribution imaging
The MC38-bearing mice were injected with 100 mg/kg of [10B]BPA by the tail vein. Then, the tumour tissue was dissected one hour later. The tissues were flash-frozen in liquid nitrogen and then rewarmed for 1 h at −20°C. The section thickness was set to 14 μm. Spatial imaging of boron was conducted via matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS, Bruker) at a resolution of 50 μm.

X-ray radiation delivery
X-ray irradiation was conducted via an X-ray generator (RS2000 Pro 225, 225 kV, 17.7 mA; Rad Source Technologies). The cells received a total dose of 10 Gy at a rate of 2.09 Gy/min. For animal irradiation, mice anaesthetized with tribromoethanol were secured onto a module while the remainder of the body was shielded with 5-mm-thick lead. The tumours were irradiated locally at a dose of 4 Gy at a rate of 2.09 Gy/min.

Extracellular ATP assay
The treated cells were digested and transferred to a 96-well plate. Following the manufacturer’s instructions, the extracellular ATP level was measured via the Real Time-Glo Extracellular ATP Assay (GA5010, Promega).

Cell viability assays by CCK-8
To evaluate cell viability following BNCT treatment, MC38 and B16F10 cells were incubated with [10B]BPA overnight. The cells were then irradiated with thermal neutrons for 5 min or under 4 Gy X-ray irradiation. After treatment, MC38 and B16F10 cells were seeded in 96-well plates at a concentration of 5×10³ cells per well. Cell viability was assessed via the CCK-8 (C0040, Beyotime) assay at 24, 48-, 72-, 96-, and 120 h post-irradiation, following the manufacturer’s protocol. For the combination of BNCT with inhibitors, MC38 and B16F10 cells were incubated overnight with [10B]BPA and/or various inhibitor molecules. The inhibitors included Necrostatin-1s (20 μM, HY-14622A, MCE), GSK872 (5 μM, HY-101872, MCE), Z-VAD-FMK (40 μM, HY-16658B, MCE), Z-IETD-FMK (25 μM, HY101297, MCE), ZDEVD-FMK (50 μM, HY-12466, MCE), and Ferrostatin-1 (5 μM, HY-100579, MCE). The cells were subsequently irradiated with thermal neutrons for 5 min.

H&E staining
Tumours were excised from the mice in different groups 1, 3, and 5 days after treatment. The tumour tissues were fixed in 4% paraformaldehyde (P0099, Beyotime), embedded in paraffin, sectioned into slices, and stained with hematoxylin and eosin (C0105S, Beyotime). The slices were photographed via 3D histech, and the data were analyzed with ImageJ (version 1.53a).

Transmission electron microscopy imaging
Transmission electron microscopy (TEM) images were acquired using a JEM-2100F microscope (JEOL Ltd., Japan). The sample preparation procedure was as follows: cells were initially fixed at room temperature for ~5 min in 2.5% glutaraldehyde fixative. The cells were then gently scraped in one direction using a cell scraper, and transferred into a centrifuge tube and centrifuged for 2 min. After discarding the supernatant, fresh fixative was added, and the samples were further fixed at room temperature for 30 min in the dark.

Western blot analysis
Western blot analysis was used to detect phospho-γH2AX in BNCT-treated or X-ray-treated MC38 cells in vitro. A total protein extraction kit (BC3710, Solarbio) was used to extract proteins from the tumours. The concentrations were determined via the BCA protein assay kit (PC0020, Solarbio). Proteins were diluted with 5× SDS-PAGE buffer, boiled for 15 min, subjected to appropriate concentrations for electrophoresis, and then transferred to PVDF membranes. The primary antibodies used were specified as follows: GAPDH (D16H11) XP Rabbit monoclonal mAb (5174S, CST, 1:200), Phospho-histone H2A.X (Ser139) (20E3) (9718S, CST, 1:200). The secondary antibody used was anti-rabbit (7074S, CST). Proteins were analyzed via the GelDoc Go Gel Imaging System (Bio-Rad).

Cell sorting and quantitative PCR
The MC38 and B16F10 tumours were digested into single cells, followed by staining with anti-mouse CD45 FITC (147710, BioLegend) on ice for 1 h. The immune cells (CD45+) and non-immune cells (CD45-) were sorted using a flow cytometer (BD FACSAria III Cell Sorter). RNA was extracted from the sorted cells using RNeasy Mini Kit (74104, QIAGEN), and then cDNA was subsequently synthesized via a PrimeScript RT reagent Kit (RR037A, TaKaRa). The expression level of the Slc7a5 gene was measured via quantitative PCR (qPCR) using LightCycler 96 System (Roche) and BlazeTaq SYBR Green qPCR Mix 2.0 (QP041, GeneCopoeia). Primers were synthesized by Rui Biotech. qPCR was performed for 30 s at 95°C, 45 cycles at 95°C for 10 s and 60°C for 30 s, with subsequent melting-curve analysis. The expression of target genes was normalized to that of β-actin. The primers used for qPCR with reverse transcription were as follows: β-actin (forward 5′-GTGACGTTGACATCCGTAAAGA-3′, reverse 5′-GCCGGACTCATCGTACTCC-3′), Slc7a5 (forward 5′-CTGACACCTGTGCCATCACT-3′, reverse 5′-TTCACCTTGATGGGACGCTC-3′).

Single-cell sample preparation
B16F10 tumours: Three tumour-bearing mice were irradiated with 4 Gy X-ray. Three tumour-bearing mice were intravenously injected with 100 mg/kg of [10B]BPA, and one hour later, tumours were irradiated with 30 min of thermal neutron irradiation. Three tumour-bearing mice were left untreated. Tumourswere harvested one or seven days after treatment. MC38 tumours: Three tumour-bearing mice were irradiated with 4 Gy X-ray. Three tumour-bearing mice were intravenously injected with 100 mg/kg of [10B]BPA, and 1 h later, the tumours were irradiated with 20 min thermal neutron irradiation. Three tumour-bearing mice were left untreated. Tumours were harvested 5 days after treatment. The tumours were digested into single cells. Three tumours from the same group were combined into one sample. For B16F10 tumours 7 days after treatment, cells were stained with anti-mouse CD45 FITC (147710, BioLegend) and sorted via flow cytometry (BD FACSAria III Cell Sorter). CD45+ immune cells were enriched at a ratio of CD45- to CD45+ of 1:2. Libraries were constructed using the Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 (10x Genomics), and immune repertoire measurements were conducted via the Chromium Single Cell V(D)J Reagent Kit (10x Genomics) following the manufacturer’s instructions. Libraries were sequenced as paired-end 150-base reads on an Illumina HiSeq-X platform.

Single-cell RNA-seq data preprocessing
Here, we performed analyses of the single-cell sequencing data derived from MC38 (mouse-derived) and B16F10 (mouse-derived) samples separately. Newly generated single-cell sequencing data were aligned with the mm10 mouse reference genome and quantified via Cell Ranger (version 7.1, 10x Genomics Inc.). The cells with fewer than 200 genes detected and a mitochondrial unique molecular identifier (UMI) count percentage larger than 10% were filtered out. The genes detected in less than three cells were also filtered out. To remove the potential doublets, Scrublet53 was used for each sample with the expected doublet rate set to be 0.05, and cells with the predicted doublet Score larger than 0.3 were further filtered out. We next normalized the count data using the normalize_total function with the default parameter in Scanpy54. All the normalized data were logarithmically transformed for downstream analyses. Highly variable genes (HVGs) were selected, and principal component analysis (PCA) was performed on the matrix of HVGs to reduce noise using the scanpy.tl.pca function. The batch effects were corrected by the Harmony55 algorithm, in which the parameter of the scanpy.external.pp.harmony_integrate function was set to “batch_key=’sample”. The nearest neighbours distance matrix was calculated with the scanpy.pp.neighbors function and Uniform Manifold Approximation and Projection (UMAP) was employed for visualization via the scanpy.tl.umap function with default parameters. Using the scanpy.tl.leiden function, we performed two rounds of unsupervised clustering to reveal the structure of whole-cell and single-cell populations, such as cancer cell and T-cell populations.

Single-cell RNA-seq data cell-type annotation
To define the main cell types, we categorized the cells according to commonly used cell markers: Col1a2 for fibroblasts, Pecam1 for endothelial cells, Ptprc for immune cells, Cd8a for CD8+ T cells, Cd4 for CD4+ T cells, Klrk1 for NK cells, Cd79a for B cells, Itgam for myeloid cells, Fcn1 for monocytes, Cd68 for macrophages, and H2-Ab1 for dendritic cells. Specifically, to identify the cancer cells, we used Mlana and Pmel for the B16 tumours and Epcam for the MC38 tumours. Furthermore, the infercnvpy Python package was used to infer somatic Copy Number Variation (CNV) in which fibroblast, endothelial, and immune cells were labelled with a normal background for calling CNVs.

Differential gene expression and pathway enrichment analysis
We performed differential gene expression analysis of different cellular populations by using scanpy.tl.rank_genes_groups. Next, we performed the pathway enrichment analysis of those differentially expressed genes using the enricher function of cluster-Profiler V4.6.256 (Gene Ontology genesets or cancer hallmark genesets from MSigDB).

Calculation of the signature score and definition of genesets
We used the sc.tl.score_genes to calculate the signature score of a specific geneset. The antigen processing and presentation geneset was obtained from the GO:0030333 of Gene Ontology. The MHC class 1-related geneset was defined as H2-K1, H2-Ke6, H2-D1, H2-Q4, H2-T23, and H2-T22. The activation and exhaustion genesets of CD8 T+ cells were obtained from a previous study that uncoupled the dysfunction and activation gene modules in tumour-infiltrating T cells57. The natural killer cell-mediated cytotoxicity geneset was obtained from the GO:0042267 of Gene Ontology.

TCR analysis
The TCR sequences for each single cell, derived from 10x Genomics data, were processed via Cell Ranger (version 7.1) with the manufacturer-provided mouse V(D)J reference genome. For all assembled TCR contigs, we excluded those that had low-confidence, were nonproductive, or had UMIs < 2. A TCR clone was defined as a combination of the same TCR α chain and TCR β chain, sharing identical V and J gene segments and the same CDR3 amino acid sequence. TCR clones with more than two cells were classified as expanded.

Cell cycle analysis
At the designated time points, the adherent cells were harvested post-treatment. The Propidium Iodide Flow Cytometry Kit (ab139418, Abcam) was used to distinguish cell cycle stages on basis of the cellular DNA content, following the manufacturer’s protocol.

Immunofluorescence
Tumour tissues were prepared as cryostat sections. These sections were fixed with 4% paraformaldehyde and treated with a permeabilization solution containing 0.5% Triton X-100 for 30 min at room temperature, and then stained with the following primary antibodies: phospho-histone H2A.X (Ser139) antibody (9718S, CST, 1:200, clone number 20E3), anti-HMGB1 polyclonal antibody (ab18256, Abcam; 1:200), anti-Calreticulin polyclonal antibody (ab2907, Abcam, 1:200) or CD8a monoclonal antibody (14-0081-82, Invitrogen; 1:200, clone number 53-6.7), as well as the secondary antibody anti-rabbit IgG (H+L) (4412S, CST; 1:1000). The samples were mounted with anti-fluorescence quenching sealing tablets (G1407, Servicebio) containing 4’,6-diamidino-2-phenylindole (DAPI) and then imaged using a fluorescence microscope (IX73, Olympus). The visual fields were randomly selected and analyzed via ImageJ (version 1.53a).

Multiplex IHC staining was performed on 4-mm-thick, formalin-fixed, paraffin-embedded slides using an Opal multiplex IHC system (20241001 kit, Lifty) according to the manufacturer’s instructions. After slide preparation and heat-induced epitope retrieval, the slides were blocked with Lifty Antibody Diluent Block buffer. The following primary antibodies were used: anti-CD8 monoclonal antibody (ab217344, Abcam, clone number EPR21769), anti-CD11c monoclonal antibody (ab219799, Abcam, clone number EPR21826), anti-MHC class II monoclonal antibody (14-5321-82, Invitrogen, clone number M5/114.15.2), and anti-F4/80 monoclonal antibody (70076T, CST, clone number D2S9R). Each slide was baked in the oven at 75°C for 1 h. Then, the slides were deparaffinized with xylene and rehydrated through a graded series of ethanol solutions. After antigen retrieval in a microwave, the slides were washed with TBST wash buffer. After blocking, the sections were incubated with primary antibodies for 1 h and then incubated with the polymer HRP Ms+Rb as the secondary antibody for 10 min at room temperature. Opal fluorophores were pipetted onto each slide for 10 min at room temperature, and the slides were microwaved to strip the primary and secondary antibodies. We then repeated the same protocol using the next primary antibody target. Finally, at room temperature, DAPI was pipetted onto each slide for 10 min. The slides were covered with Lifty-BD-FM-1, and images were taken using an AKOYA automated quantitative pathology system. The images were analysed by inForm 2.8.0 software (Akoya Biosciences, Inc., USA).

Enzyme-linked immunosorbent assay (ELISA)
Blood was collected via the ocular method, and ELISA assays for IFN-α, IFN-β, and IFN-γ were performed on mouse serum using LumiKine Xpress mIFN-α 2.0 (luexmifnav2, Invitrogen), LumiKine Xpress mIFN-β 2.0 (luexmifnav2, Invitrogen), and the IFN-γ pre-coated ELISA kit (430807, BioLegend) according to the manufacturer’s instructions.

BMDCs and T cells co-culture
BMDCs were generated by culturing bone-marrow cells in the presence of 20 ng/mL recombinant murine GM-CSF (315-03-250 μg, PeproTech) and 200 ng/mL recombinant murine Flt3 (250-31L-250 μg, PeproTech) for 7 days. Then the BMDCs were treated with B16F10 cells in transwell (140652, Thermo Fisher Scientific) with 2.5×10⁵ BMDCs and 5×10⁴ tumour cells per well. After 72 h incubation, cells were sorted, and surface markers CD80/86 were analysed on flow cytometry using anti-mouse CD80 Brilliant Violet 650 (104732, BioLegend), anti-mouse CD86 PE (105007, BioLegend). OTI CD8+ T cells were isolated from spleens using CD8+ T cell isolation kit (130-104-075, Miltenyi Biotec). T cells were then co-cultured with 500 μg/mL OVA (A5503, sigma) and BMDCs at 8:1. After 72 h incubation, T cells were analysed after staining with anti-mouse CD3-PE (100206, BioLegend), anti-mouse CD25-PE/Cyanine7 (102016, BioLegend), and anti-mouse CD69-BV421 (104545, BioLegend) by flow cytometry.

Flow cytometry
Tumour tissues were dissociated as described above to obtain single-cell suspensions for detecting cell viability (immune cells and non-immune cells), T cells, and dendritic cells. The cells were labelled with the Zombie Aqua Fixable Viability Kit (423102, BioLegend) in PBS and blocked with anti-mouse CD16/32 (101302, BioLegend). Surface staining was performed on ice for 30 min, followed by a wash with FACS buffer (PBS+2% FBS) and resuspension in FACS buffer for analysis on a BD LSR Fortessa X-20 Cell Analyzer. All the data were analyzed using FlowJo (version 10). To assess the viability of immune cells or non-immune cells in tumours treated with or without BNCT, single-cell suspensions were stained with anti-mouse CD45 FITC (147710, BioLegend). For DCs, single-cell suspensions were stained with the following anti-mouse antibodies against: anti-mouse CD45 FITC (147710, BioLegend), anti-mouse I-A/I-E Brilliant Violet 421 (107632, BioLegend), anti-mouse CD11c APC (117310, BioLegend), anti-mouse Ly-6C PerCP/Cyanine 5.5 (128012, BioLegend), anti-mouse F4/80 Alexa Fluor 700 (123130, BioLegend), anti-mouse CD80 Brilliant Violet 650 (104732, BioLegend), anti-mouse CD86 PE (105007, BioLegend). For T cells, the following antibodies were used: anti-mouse CD3 PE (100206, BioLegend), anti-mouse CD4 FITC (100510, BioLegend), and anti-mouse CD8a APC (100712, BioLegend). For memory cells in mouse blood, spleen, and lymph nodes, the single-cell suspensions obtained were processed similarly and stained with the following anti-mouse antibodies against: anti-mouse CD3 PE (100206, BioLegend), anti-mouse CD4 FITC (100510, BioLegend), anti-mouse CD8a APC (100712, BioLegend), anti-mouse/human CD44 Brilliant Violet 605 (103047, BioLegend), and anti-mouse CD62L Brilliant Violet 421 (104436, BioLegend). The catalogue numbers of the antibodies used in this study are shown in Supplementary Table 1.

Statistical analysis
The data are shown as the mean ± SEM. Statistical analyses were performed with GraphPad Software (version 10).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors showed that BCNT treatment showed a gene expression profile that caused G1 arrest compared to x-ray treatment by the single-cell RNA-seq. They also show upregulation of immune-related genes and cytokine expression, and functional enhancement of DCs within the tumour, by BCNT treatment.\nHowever, it is difficult to say that the paper is organized to allow the reader to distinguish between what was previously known and what was newly revealed. For example, I think the early section on results should be in the introduction, and some of the explanation of the results should be in the discussion. Furthermore, the discussion does not cite any references, which is a structure I have never seen in a paper before and is difficult to understand.\nFurthermore, there are no citations in this manuscript that seem necessary; for example, Mishima et al Strahlenther Onkol 1989 or Wongthai et al Cancer Sci 2015. on BPA-mediated BNCT should be cited.\nI strongly recommend that authors re-examine their paper and rewrite it.", "reviewer_2": "This study investigates the efficacy of boron neutron capture therapy (BNCT) in pre-clinical model of melanoma (BF16F10 cells) and colorectal cancer (MC38 cells), in comparison with “standard” X-ray treatment. In particular, it investigates the effect of this radiotherapy (RT) on the immune response.\nMain claims of this study are that BNCT a) minimally affects immune cell viability and functionality in the tumour microenvironment; b) triggers immunogenic cell death and consequently promotes an immune-mediated anti-tumour response; c) enhances DC activity, T cell activity and NK cell cytotoxicity.\nBNCT is a form of RT that has gained increasing attention in recent years, following its approval for clinical use in cancer treatment. However, the downstream mechanisms triggered by BNCT in target tissues remain poorly understood. In particular, the broader effects of BNCT on the immune system, and whether these could promote (some sort of) abscopal effect, are largely unknown. In this context, the present study is extremely timely and presents novel findings that may support the broader application of BNCT across cancer types.\nThat said, some of the study's conclusions would benefit from additional mechanistic validation, particularly those related to BNCT-induced immunogenic cell death and the causal link between dendritic cell (DC) activation and adaptive immune responses.\n- A central finding of the study is that BNCT induces immunogenic cell death, which may explain the differential outcomes observed compared to conventional X-ray radiotherapy. It is therefore surprising that comparative data with X-ray RT are missing from several panels in Fig.2 (2a-b-c,h-1,l-m) and Ext.Fig.3 (3g-h,i-j). Is there any specific reason for this? Including these data would strengthen the conclusions, for instance in relation to interferon responses following irradiation.\n- The authors speculate that BNCT may induce necrosis or a form a caspase independent cell death (CICD), after excluding a number of known mechanisms. Again, as this is a major mechanistic claim, additional data demonstrating inflammatory signalling downstream of BNCT would be valuable. For instance, the authors could assess markers of MOMP (mitochondria outer membrane permeabilization), that was proven to activate NF-kB and cGAS-STING pathways (https://doi.org/10.1038/s41418-017-0012-4 and https://doi.org/10.1038/ncb3596).\n- One of the advantages of BNCT is the selective targeting of tumour cells, attributed here to differential expression of Slc7a5. While Figure 3 suggests that Slc7a5 expression is lower in the immune compartment, its expression across other non-malignant stromal cells (e.g., fibroblasts, endothelial cells) is not addressed. Mining scRNA-seq data for Slc7a5 expression across these populations, as well as across the various tumour cell clusters, would add valuable context. Additionally, it would be important to assess Slc7a5 expression across specific immune cell subsets to support the claim of immune sparing.\n- The sparing and activation of local immunity is posited as a distinctive advantage of BNCT over standard X-ray. However, Figure 3i–m does not include a comparison to X-ray treatment, which limits the strength of this conclusion.\n- The link between dendritic cell activation and increased T or NK cell cytotoxicity is inferred solely from in silico analyses. Supporting this with functional ex vivo assays would considerably strengthen the claim. For example, if numbers are sufficient, testing DC-mediated activation of T or NK cells ex vivo would provide mechanistic evidence for the proposed pathway.", "reviewer_3": "This study demonstrates that boron neutron capture therapy (BNCT), a high-precision radiotherapy, selectively kills tumor cells while sparing immune cells in the tumor microenvironment. Using murine models (B16F10 and MC38), the authors show that BNCT induces immunogenic cell death, enhances dendritic cell activation, expands CD8⁺ T cell populations, and promotes systemic anti-tumor immunity with long-term memory. Single-cell RNA sequencing revealed inflammatory transcriptional changes and increased antigen presentation post-BNCT. Functional experiments confirmed that CD8⁺ T cells are essential for sustained tumor control. These findings support BNCT as an immune-sparing modality that synergizes with anti-tumor immunity.\nMajor Issues\n1. While BNCT is very effective in destroying tumors, the X-ray dose being compared is 4 Gy, which is too low to be clinically effective. It is unclear whether the difference in immune activity is due to “immune cell preservation” or simply “difference in the amount of tumor destruction. Comparative experiments with matched dose effects are needed.\n2. There is a difference in the treatment process between the BNCT and X-ray groups. The BNCT group receives BPA administration, anesthesia, and prolonged beam irradiation, whereas the X-ray group does not include these elements and their effect on the immune response cannot be ruled out. Sham administration and anesthesia procedures must also be applied to the x-ray group.\n3. Cells derived from 3 mice in each group were pooled for analysis and may be considered n=1 for statistical purposes. Independence of statistical analysis is not ensured and generalization of results requires careful interpretation.\n4. BNCT-treated tumor cells have been shown to be effective as a vaccine, but have not been compared to X-ray-treated cells, an important control group to support the claim that BNCT induces more immunogenicity.\n5. The analysis time points are not aligned with Day 1/7 for B16F10 and Day 5 for MC38, which may cause bias in the comparison. The difference in time points needs to be clearly explained.\n6. There is an increase in the percentage of immune cells in the tumor after BNCT treatment, but this may be a “relative increase” due to a decrease in tumor cells rather than an increase in the number of immune cells. Absolute count or density data should be presented.\n7. ATP, HMGB1, and IFN have been evaluated, but ICD indicators such as surface calreticulin have not been evaluated. Complementary discussions will provide a better understanding of ICD induction by BNCT.\n8. While NK cell activity has been suggested by scRNA-seq, functional verification (e.g., NK cell removal experiments) has not been performed. It would be more comprehensive if added to the discussion as a future issue.\n9. Single-cell RNA-seq and TCR data are not publicly available and insufficient in terms of reproducibility; registration with GEO and ArrayExpress and publication of analysis codes on GitHub are desired.\nMinor Issues\n• There are several typos, including “gliomas” for “gliomass” in line 76, “themice” for “the mice” in line 590, and “filte” in line 595. They need to be corrected.\n• Figure reference formatting: In one instance, a duplicate reference is present: “Extended Data Fig. Fig. 2a,b” has “Fig.” written twice. This appears to be a minor editorial slip. (Line 121)"}, "author_response": "Dear reviewers,\nOn behalf of the authorship group, I would like to thank you for taking the time to evaluate our\nmanuscript. We are deeply grateful for your appreciation of our work. Please kindly find our point-\nby-point response to the reviewers’ comments below. Most of the panels in Figures for Response\nhave been edited into the appropriate section of the article or Extended Data.\nReviewer #1\nThe authors showed that BCNT treatment showed a gene expression profile that caused G1 arrest\ncompared to x-ray treatment by the single-cell RNA-seq. They also show upregulation of immune-\nrelated genes and cytokine expression, and functional enhancement of DCs within the tumour, by\nBCNT treatment.\nHowever, it is difficult to say that the paper is organized to allow the reader to distinguish between\nwhat was previously known and what was newly revealed. For example, I think the early section on\nresults should be in the introduction, and some of the explanation of the results should be in the\ndiscussion. Furthermore, the discussion does not cite any references, which is a structure I have never\nseen in a paper before and is difficult to understand.\n- We sincerely thank the reviewer for this valuable suggestion. Following the reviewer’s advice, we\nhave reorganized the introduction to provide a clearer distinction between what was already known\nand what is newly revealed in our work. And the background information of early section on results\nhave been moved to the introduction to provide better context. In the Discussion, we expanded the\ninterpretation of our findings, and incorporated relevant literature citations. These changes improve\nthe clarity and logicality of the manuscript and better highlight the novelty of our work.\nThe following are the details of the modification:\n1) In the revised version of introduction, we first summarized the current understanding of how\nconventional radiotherapy modulates the tumour immune microenvironment, highlighting the\ndilemma between tumour ablation and immune cell preservation as a critical unsolved problem. We\nthen introduced BNCT by presenting its physical principles and its theoretical positioning as a single-\ncell precision radiotherapy strategy, emphasizing that immune cell sparing has largely remained a\nhypothesis without direct biological validation. Next, we discussed the encouraging clinical outcomes\nof BNCT. However, there has been a lack of immunological exploration of the favorable clinical\noutcomes associated with long-term survival. Then we clearly stated how our study addresses these\ngaps: by providing direct in vivo experimental evidence of immune cell sparing, and by elucidating\nthe immune mechanisms that support durable systemic anti-tumour responses.\n2) In the revised version of discussion, we first introduced the conceptual framework of single-cell\nradiotherapy and then demonstrated how our experimental data validate this paradigm through BNCT.\nWe expanded the scope to highlight that BNCT could not only passively preserve immune cells but\nalso actively induce systemic immune responses. We further integrated tumour heterogeneity into the\ndiscussion and used scRNA-seq to dissect distinct tumour subpopulations with different fates.\nBeyond tumour cells, we also incorporated new evidence on immune microenvironment remodeling,\nsuch as dendritic cell activation, CD8+ T cell function, the expansion of the TCR repertoire, and NK\ncell cytotoxicity. In addition, we identified unresolved mechanistic questions, particularly the precise\ndefinition of BNCT-induced cell death, pointing to possible pathways for future study. Finally, we\nsummarized the value of single-cell radiotherapy, which may offer a transformative pathway to\noptimize therapeutic outcomes.\nFurthermore, there are no citations in this manuscript that seem necessary; for example, Mishima et\nal Strahlenther Onkol 1989 or Wongthai et al Cancer Sci 2015. on BPA-mediated BNCT should be\ncited. I strongly recommend that authors re-examine their paper and rewrite it.\n- We sincerely appreciate the reviewer’s careful reading of our manuscript and for pointing out the\nabsence of key references. We fully agree that the pioneering work of Mishima et al. (Strahlenther\nOnkol, 1989) and the mechanistic study by Wongthai et al. (Cancer Sci, 2015) are essential to provide\na more complete context for BPA-mediated BNCT. The report by Mishima and colleagues represents\nthe first human clinical trial of BNCT for melanoma, which demonstrated the potential therapeutic\nvalue of this modality. Wongthai and co-workers made an important contribution by clarifying the\nrole of amino acid transporters, particularly LAT1, in mediating the uptake of BPA into tumour cells.\nIn addition to these two essential references, we have also re-examined the entire manuscript to\nidentify other areas where additional citations could enhance the rigor and context of our work.\nFollowing your constructive advice, we have re-examined our manuscript carefully and revised the\nrelevant sections. We are grateful for this helpful suggestion, and we apologize for the oversight in\nthe original submission.\nReviewer #2\nThis study investigates the efficacy of boron neutron capture therapy (BNCT) in pre-clinical model\nof melanoma (BF16F10 cells) and colorectal cancer (MC38 cells), in comparison with “standard” X-\nray treatment. In particular, it investigates the effect of this radiotherapy (RT) on the immune response.\nMain claims of this study are that BNCT a) minimally affects immune cell viability and functionality\nin the tumour microenvironment; b) triggers immunogenic cell death and consequently promotes an\nimmune-mediated anti-tumour response; c) enhances DC activity, T cell activity and NK cell\ncytotoxicity.\nBNCT is a form of RT that has gained increasing attention in recent years, following its approval for\nclinical use in cancer treatment. However, the downstream mechanisms triggered by BNCT in target\ntissues remain poorly understood. In particular, the broader effects of BNCT on the immune system,\nand whether these could promote (some sort of) abscopal effect, are largely unknown. In this context,\nthe present study is extremely timely and presents novel findings that may support the broader\napplication of BNCT across cancer types.\nThat said, some of the study's conclusions would benefit from additional mechanistic validation,\nparticularly those related to BNCT-induced immunogenic cell death and the causal link between\ndendritic cell (DC) activation and adaptive immune responses.\n- We thank the reviewer for enthusiastic comments on the novelty and suggestions for further\ndevelopment of our work.\n1) A central finding of the study is that BNCT induces immunogenic cell death, which may explain\nthe differential outcomes observed compared to conventional X-ray radiotherapy. It is therefore\nsurprising that comparative data with X-ray RT are missing from several panels in Fig.2 (2a-b-c,h-\n1,l-m) and Ext.Fig.3 (3g-h,i-j). Is there any specific reason for this? Including these data would\nstrengthen the conclusions, for instance in relation to interferon responses following irradiation.\n- We sincerely thank the reviewer for this insightful comment. We apologize for the omission of\ncomparative data with X-ray radiation in several panels. Following the reviewer’s suggestion, we\nhave included the corresponding data (Fig. R1–5). These additional results indeed help to better\nhighlight the unique advantages of BNCT. In particular, the comparison demonstrates that BNCT\ninduces more robust immunogenic cell death, more irreparable damage, and triggering long-term\ncycle arrest than conventional X-ray treatment, further supporting our conclusion on the\nimmunological effects of BNCT.\nThe data including the X-ray group is as follows:\nFig. R1 Corresponding to Figure 2a–c, cell morphology of B16F10 or MC38 cells. Compared with the X-ray\ngroup, BNCT exhibited stronger tumour cell–killing ability.\nFig. R2 Corresponding to Figure 2h–i, extracellular ATP release. Within 120 hours, X-ray treatment did not\ninduce a notable change in ATP release compared with the untreated control. In contrast, the BNCT group\nshowed a significant increase in extracellular ATP levels between 60 and 84 hours in both B16F10 and MC38\ncell lines.\nFig. R3 Corresponding to Figure 2l–m, serum IFN-α, IFN-β, and IFN-γ levels. In the “cold” B16F10 tumour\nmodel, interferon levels in mouse serum showed an upward trend after BNCT compared with X-ray treatment,\nbut the difference was not statistically significant. In the “hot” MC38 tumour model, BNCT treatment resulted\nin a significant increase in serum IFN-α and IFN-β compared with X-ray treatment. Together with HMGB1\nand extracellular ATP release, these results further indicate the superior ability of BNCT to induce\nimmunogenic cell death.\nFig. R4 Corresponding to Extended Data Figure 3f–h, dynamic changes in the cell cycle. After X-ray treatment,\nthe proportion of cells in G2/M phase increased, peaking at approximately 50% at 12 hours post-irradiation,\nand cell cycle distribution stabilized by 72 hours, suggesting that cells had largely repaired radiation-induced\ndamage. In contrast, following BNCT treatment, over 60% of cells were in G2/M phase at 24 hours, and cell\ncycle disruption persisted up to 96 hours. These data indicated that BNCT induced a more prolonged cell cycle\narrest, leading to a stronger tumour cell killing effect than X-ray irradiation.\nFig. R5 Corresponding to Extended Data Figure 3i–l, exploration of cell death pathways. The inhibition of\ncaspase proteins did not prevent BNCT-induced cell death, whereas pan-caspase inhibitors partially rescued\nsome cells exposed to X-ray irradiation. This indicated that caspases play a key role in X-ray induced cell\ndeath, while BNCT-mediated cytotoxicity is largely caspase-independent.\n2) The authors speculate that BNCT may induce necrosis or a form a caspase independent cell death\n(CICD), after excluding a number of known mechanisms. Again, as this is a major mechanistic claim,\nadditional data demonstrating inflammatory signaling downstream of BNCT would be valuable. For\ninstance, the authors could assess markers of MOMP (mitochondria outer membrane\npermeabilization), that was proven to activate NF-kB and cGAS-STING pathways\n(https://doi.org/10.1038/s41418-017-0012-4 and https://doi.org/10.1038/ncb3596).\n- We sincerely thank the reviewer for raising this important mechanistic point and for providing\nvaluable references. Mitochondrial outer membrane permeabilization (MOMP), allowing the release\nof proteins like cytochrome c, typically triggering apoptosis, it has also been shown to drive caspase-\nindependent cell death (CICD)1. As suggested, we investigated whether BNCT-induced cell death\ninvolves MOMP1-3.\nWe first assessed mitochondrial morphology after BNCT. Electron microscopy revealed\nmitochondrial vacuolization and cristae disorganization after BNCT treatment (Fig. R6), consistent\nwith mitochondrial perturbation.\nFig. R6. a,b, Representative transmission electron microscopy (TEM) images of B16F10 tumour cells (a) and\nMC38 tumour cells (b). Scale bar, 1 μm.\nMOMP leads to the dissipation of the mitochondrial membrane potential4. JC-1 dye is commonly\nused to assess mitochondrial membrane potential; when the membrane potential decreases, JC-1\ntransitions from its aggregate form to a monomer5. JC-1 staining revealed a reduction in\nmitochondrial membrane potential following BNCT treatment, suggesting mitochondrial\nperturbations (Fig. R7).\nFig. R7. a-c, JC-1 aggregate to monomer ratio of MC38 tumour cells at 72 h post-treatment (a), B16F10\ntumour cells at 72 h post-treatment (b), B16F10 tumour cells at 120 h post-treatment (c). n = 4 independent\nreplicates for each group tested, two-tailed unpaired Student’s t-test, ****p < 0.0001.\nAt the transcriptional level, we examined the expression of MOMP-related genes, including BBC3\n(PUMA), BCL2L11 (BIM), PMAIP1 (NOXA), and BAX6 (Fig. R8), which are classical BCL-2 family\nmembers. The proteins transcribed from these genes have been reported to act as transducers of\ncellular stress signals and regulate mitochondrial permeability. Among these, BAX expression\nshowed the most pronounced upregulation after BNCT, consistent with its well-recognized role in\nMOMP initiation due to its ability to form pores in the outer mitochondrial membrane7.\nFig. R8. a, b, The expression of MOMP-related genes of MC38 tumour cells (a) and B16F10 tumour cells (b).\nThese results suggested that BNCT induced MOMP and upregulated the mRNA expression of Bax.\nTo directly investigate the role of Bax in BNCT induced cell death, we employed BAX knockout cell\nlines (Fig. R9). However, BNCT-induced cell death was only partially rescued in BAX-deficient\ncells, with viability remaining at ~42% after 120 hours, indicating that while mitochondrial damage\noccurs, MOMP is not the sole or decisive mechanism driving cytotoxicity.\nFig. R9. Cell viability of A549 cells after treatment at different time points, as detected by a CCK-8 assay (n\n= 6 independent replicates for each group).\nIn addition, because MOMP can trigger inflammatory signaling through release of mitochondrial\nDNA and subsequent activation of cGAS–STING and NF-κB pathways8, we further explored these\npossibilities. Bioinformatic analyses (Fig. R10 a,b) and functional validationusing STING-knockout\ntumour cells (Fig. R10 c) did not reveal a major contribution of cGAS–STING signaling to BNCT\nresponse. Similarly, NF-κB pathway activation was not observed in MC38 tumour cells following\nBNCT (Fig. R11). Taken together, our results demonstrate that although BNCT induces\nmitochondrial damage consistent with MOMP, cell death is not fully dependent on this pathway, nor\non cGAS–STING or NF-κB signaling.\nFig. R10. a,b, cGAS-STING signature in B16F10 tumour cells (a) and MC38 tumour cells (b). Top and bottom\nquartiles are shown with horizontal lines at the median, and whiskers denote the range of the data that falls\nwithin 1.5 times the interquartile range above the third quartile and below the first quartile. c, Average tumour\nvolumes of each group of STING-knockout MC38 bearing mice. n = 6 mice.\nFig. R11. NF-κB signature in MC38 tumour cells. Top and bottom quartiles are shown with horizontal lines\nat the median, and whiskers denote the range of the data that falls within 1.5 times the interquartile range above\nthe third quartile and below the first quartile.\nIn our study, pan-caspase inhibition failed to rescue BNCT-induced cell death, indicating that\ncaspase-independent mechanisms may be involved. Combined with the results of our supplemental\nexperiment, thses findings suggest BNCT may trigger multiple overlapping cell death pathways that\ncannot be rescued by blocking a single typical pathway. Therefore, although we cannot conclusively\nattribute the upregulation of ICD markers and cytokines in tumour cells after BNCT to the classic\nsignaling pathways, our data help to rule out a predominant role of classical apoptosis or caspase-\ndependent mechanisms, and instead support the possibility of caspase-independent modes of cell\ndeath. We recognize this limitation and have added further discussion of this issue in the revised\nmanuscript. This point indeed opens a valuable direction for future investigation, and we sincerely\nthank the reviewer again for this constructive suggestion.\n3) One of the advantages of BNCT is the selective targeting of tumour cells, attributed here to\ndifferential expression of Slc7a5. While Figure 3 suggests that Slc7a5 expression is lower in the\nimmune compartment, its expression across other non-malignant stromal cells (e.g., fibroblasts,\nendothelial cells) is not addressed. Mining scRNA-seq data for Slc7a5 expression across these\npopulations, as well as across the various tumour cell clusters, would add valuable context.\nAdditionally, it would be important to assess Slc7a5 expression across specific immune cell subsets\nto support the claim of immune sparing.\n- We sincerely thank the reviewer for raising this important point. To further address the issue, we\nexamined the expression of Slc7a5 in non-malignant cell types. Using qPCR, we compared multiple\nnon-tumour cells with MC38 and B16F10 tumour cells (Fig. R12a). We found that Slc7a5 expression\nwas consistently lower in non-tumour cells, including fibroblast, endothelial cells, epithelial cells,\nmacrophages, DCs, CD4+ T cells, CD8+ T cells, relative to tumour cells. These results suggested\nthat stromal populations may indeed express Slc7a5 at a much lower level than malignant cells.\nFig. R5. a. Relative Slc7a5 gene expression (n = 5 independent replicates for each group) of endothelial cells\n(mouse aortic endothelial cells), fibroblasts (isolated from mouse tumours), epithelial cells (mouse epidermal\nkeratinocytes), macrophages (isolated from mouse bone marrow), DCs (isolated from mouse bone marrow),\nCD4+ T cells (isolated from mouse spleen), CD8+ T cells (isolated from mouse spleen), MC38 cells, and\nB16F10 cells. b, Slc7a5 gene expression of untreated group of B16F10 tumours.\nIn parallel, scRNA-seq data analyses showed that low Slc7a5 expression across non-malignant\nstromal cells, as well as across most immune cell subsets (Fig. R12b), consistent with our wet-lab\nfindings. In contrast, tumour cells exhibited significantly higher expression. Importantly, immune\npopulations demonstrated minimal Slc7a5 expression, which supports the notion that BNCT\nselectively targets tumour cells while largely sparing immune compartments.\n4) The sparing and activation of local immunity is posited as a distinctive advantage of BNCT over\nstandard X-ray. However, Figure 3i–m does not include a comparison to X-ray treatment, which\nlimits the strength of this conclusion.\n- We thank the reviewer for this constructive comment. In response, we performed additional\nexperiments to directly compare BNCT with X-ray treatment. Using multiplex immunofluorescence\nstaining, we analyzed the distribution of immune cell subsets within the tumour tissues. As shown in\nthe Fig. R13, BNCT treatment resulted in a higher proportion of CD8+ T cells, DCs, and\nmacrophages compared with X-ray irradiation. These results provide further supported that BNCT\nnot only spares but also enhances local immune cell infiltration more effectively than conventional\nX-ray radiation.\nFig. R13. Corresponding to Figure 3i–m. a-c, Representative examples of MC38 tumours from NC group (a),\nX-ray group (b) and BNCT group (c) stained by immunohistochemical staining with anti-CD8 (green),\nanti-CD11c (purple), anti-F4/80 (brown) and anti-MHC-II (cyan) antibodies and DAPI. Scale bar, 50 μm. d–h,\nProportion of CD8+ T cells (d, *p = 0.0351, **p = 0.0037), CD11c+ myeloid cells (e, **p = 0.0014, ****p\n< 0.0001), CD11c+MHC-II+ cells (f, **p = 0.0050, ****p < 0.0001), F4/80+ macrophages (g, ****p <\n0.0001), and F4/80+MHC-II+ cells (h, NC vs X-ray, *p = 0.0230, X-ray vs BNCT, *p = 0.0141, NC vs BNCT,\n**p = 0.0042) in tumours. (n = 7 tumour sections, two-tailed unpaired Student’s t-test).\n5) The link between dendritic cell activation and increased T or NK cell cytotoxicity is inferred solely\nfrom in silico analyses. Supporting this with functional ex vivo assays would considerably strengthen\nthe claim. For example, if numbers are sufficient, testing DC-mediated activation of T or NK cells ex\nvivo would provide mechanistic evidence for the proposed pathway.\n- We are very grateful for this valuable suggestion. To provide functional evidence beyond in silico\nanalyses, we carried out ex vivo co-culture assays (Fig. R14). Specifically, B16F10 tumour cells were\ntreated with BNCT or X-ray for 3 days and then co-cultured with bone marrow–derived DCs (BMDC).\nAfter 3 days, DCs co-cultured with BNCT-treated tumour cells displayed a significantly activated\nphenotype compared with both X-ray and control groups (Fig. R14b, c). Furthermore, when these\nDCs were co-cultured with splenic CD8+ T cells, we observed clear activation of T cells within 24\nhours (Fig. R14d), confirming DC-mediated T cell stimulation.\nFig. R14. a, Treatment schemes for the co-culture assay. b and c, Expression levels of CD80 (b, NC vs X-ray,\n*p = 0.0153, X-ray vs BNCT, **p = 0.0035, NC vs BNCT, **p = 0.0017) and CD86 (c, X-ray vs BNCT, *p\n= 0.0037, NC vs BNCT, **p = 0.0055) determined by flow cytometry analysis of BMDCs. n = 3 independent\nreplicates. d, The ratio of CD25+CD69+ T cells in CD8+ T cells with co-culture with DCs under different\nconditions. n = 4 independent replicates, ****p < 0.0001, two-tailed unpaired Student’s t-test.\nRegarding NK cells, we note that NK cytotoxicity is generally not driven by the antigen-presenting\nfunction of DCs. To further test their role, we performed NK cell depletion experiments in vivo (Fig.\nR15). We found that the absence of NK cells did not sabotage the therapeutic efficacy of BNCT,\nindicating that although NK cells could be activated, they are not essential mediators of the BNCT-\ninduced anti-tumour response.\nFig. R15. Average tumour volumes (a,b) and survival fractions (c) of each group of MC38-bearing mice\ntreated with BNCT or the combination of BNCT and an anti-NK antibody (n = 5 mice).\nReviewer #3\nThis study demonstrates that boron neutron capture therapy (BNCT), a high-precision radiotherapy,\nselectively kills tumour cells while sparing immune cells in the tumour microenvironment. Using\nmurine models (B16F10 and MC38), the authors show that BNCT induces immunogenic cell death,\nenhances dendritic cell activation, expands CD8⁺ T cell populations, and promotes systemic anti-\ntumour immunity with long-term memory. Single-cell RNA sequencing revealed inflammatory\ntranscriptional changes and increased antigen presentation post-BNCT. Functional experiments\nconfirmed that CD8⁺ T cells are essential for sustained tumour control. These findings support BNCT\nas an immune-sparing modality that synergizes with anti-tumour immunity.\nMajor Issues:\n1) While BNCT is very effective in destroying tumours, the X-ray dose being compared is 4 Gy,\nwhich is too low to be clinically effective. It is unclear whether the difference in immune activity is\ndue to “immune cell preservation” or simply “difference in the amount of tumour destruction.\nComparative experiments with matched dose effects are needed.\n- We sincerely thank the reviewer for this insightful and professional comment. In our study, the\ncomparison between BNCT and X-ray was initially performed under the same absorbed dose (4 Gy).\nAs expected, BNCT demonstrated superior tumour cell killing compared with X-ray, which indeed\nraises the important question highlighted by the reviewer—whether the observed immune activation\nis primarily attributable to immune cell preservation or simply to stronger tumour destruction.\nTo address this concern, we conducted additional comparative experiments at matched biological\nequivalent doses, which in our case was defined in terms of direct tumour cell-killing effects in vitro,\nwithout the involvement of immune system. Specifically, we estimated the photon-equivalent dose\n(Gy-Eq) using established compound biological effectiveness (CBE) and relative biological\neffectiveness (RBE) factors, which are defined as the ratio of doses of a reference radiation (generally\nX-rays) to a test radiation that will produce the same biological endpoint9,10. For the melanoma model,\na CBE factor for BPA of approximately 2.1-3.39,11 and an RBE factor for beam in this work of about\n1.8 (not pubilshed). Based on dosimetric simulation, a 4 Gy absorbed dose of BNCT corresponds\napproximately to 10 Gy of X-ray, and is expected to achieve comparable tumour-killing endpoints in\nvitro.\nUsing abscopal models, we found that BNCT induced stronger anti-tumour effects than 10 Gy X-ray\nnot only in the irradiated tumours but also in the non-irradiated tumours. These results suggested that\nthe superior immune activation observed with BNCT cannot be explained solely by differences in\ntumour destruction. Instead, preservation of immune cells likely plays a critical role, contributing to\nthe enhanced systemic anti-tumour response. We appreciate the reviewer’s suggestion, which helped\nus refine our interpretation of these results.\nFig. R16. a, Treatment schemes for the abscopal effect. b,c, Average tumour volume (n = 6 mice) of each\ngroup of B16F10-bearing mice, irradiated tumours (b, ****p < 0.0001), and non-irradiated tumours (c, *p =\n0.0464). Mice were treated with 4 Gy X-ray, 10 Gy X-ray, or BNCT. d,e, Average tumour volume (n = 6 mice)\nof each group of MC38-bearing mice, irradiated tumours (d, *p = 0.0408), and non-irradiated tumours (e, *p\n= 0.0222).\n2) There is a difference in the treatment process between the BNCT and X-ray groups. The BNCT\ngroup receives BPA administration, anesthesia, and prolonged beam irradiation, whereas the X-ray\ngroup does not include these elements and their effect on the immune response cannot be ruled out.\nSham administration and anesthesia procedures must also be applied to the x-ray group.\n- We sincerely thank the reviewer for this thoughtful comment. BPA administration is an integral part\nof BNCT treatment, and therefore comparing BNCT with X-ray under these conditions necessarily\ninvolves its use. Importantly, the safety of BPA has been extensively studied, based on those applied\nin preclinical and clinical BNCT studies12-15, at the concentrations used in our experiments, it does\nnot induce immune responses. In addition, to ensure immobilization and precise tumour targeting,\nmice in the X-ray group were also anesthetized and sham administration during irradiation, and the\nanesthesia procedure was consistent between BNCT and X-ray groups. We have clarified this point\nin the revised manuscript to avoid potential misunderstanding.\n3) Cells derived from 3 mice in each group were pooled for analysis and may be considered n=1 for\nstatistical purposes. Independence of statistical analysis is not ensured and generalization of results\nrequires careful interpretation.\n- We sincerely thank the reviewer for raising this critical point regarding the statistical independence\nof our scRNA-seq samples.\nFirst, pooling cells from multiple animals into a single sequencing library was implemented to\nminimize technical batch effects, which is a major concern during our experimental design.\nProcessing each mouse individually could introduce substantial technical variation that might obscure\nthe biological signals of interest. This approach is well-established in the field; for instance, in a\nseminal study in scRNA-seq field (Cell, 2015, “Highly Parallel Genome-wide Expression Profiling\nof Individual Cells Using Nanoliter Droplets”16), researchers mixed 44,808 cells from four samples\nand then successfully identified both known neuronal classes and novel cell types.\nSecond, in scRNA-seq protocols, each cell undergoes independent RNA extraction, library\npreparation, and sequencing. Therefore, each cell can be treated as an independent observational\nunit—analogous to a sample in bulk RNA-seq. This practice is widespread and supported by\nnumerous high-impact studies. Examples include the original SMART-seq protocol paper (Nature\nBiotechnology, 2012, “Full-length mRNA-Seq from single-cell levels of RNA and individual\ncirculating tumour cells”17), a scRNA-seq atlas of colorectal cancer (Cell, 2021, “Spatially organized\nmulticellular immune hubs in human colorectal cancer”18), and, a pan-cancer T cell atlas study\n(Nature Medicine, 2023, “Pan-cancer T cell atlas links a cellular stress response state to\nimmunotherapy resistance”19), among many others.\nLastly, key findings from our scRNA-seq analysis were experimentally validated. For example, in\nFig. 5, we observed elevated antigen processing and presentation activity in DCs following BNCT\ntreatment. This was corroborated by flow cytometry, which confirmed increased expression of the\nco-stimulatory molecules CD80 and CD86 in DCs from both B16F10 and MC38 tumours after BNCT.\nThe consistency between computational and experimental results reinforces the reliability of our\nscRNA-seq findings.\nIn summary, pooling cells from multiple biological replicates is a common strategy to reduce batch\neffects in scRNA-seq studies pooling cells from multiple biological replicates is a widely adopted\nstrategy to mitigate batch effects in scRNA-seq studies. Each cell is treated as an independent unit\nduring statistical analysis—an approach supported by standard analytic protocols. The analytical\nresults were further validated by their strong concordance with follow-up experimental data.\nCollectively, these findings validate the robustness and generalizability of our statistical approach.\n4) BNCT-treated tumour cells have been shown to be effective as a vaccine, but have not been\ncompared to X-ray-treated cells, an important control group to support the claim that BNCT induces\nmore immunogenicity.\n- We apologize for the omission of comparative data with X-ray radiation in these panels. Following\nthe reviewer’s suggestion, we have included the corresponding data (Fig. R17). The results showed\nthat BNCT-treated tumour cells exhibited enhanced immunogenicity and prolonged mouse survival\ncompared with X-ray treated cells, supporting our conclusion that BNCT induces a more robust anti-\ntumour immune response.\nFig. R17. Corresponding to Figure 1i–m. a,b, Average tumour volume (a) and survival fraction (b) of each\ngroup of B16F10-bearing mice (n = 5 mice), two-tailed unpaired Student’s t-test, NC vs BNCT, **p = 0.0060,\nX-ray vs BNCT, **p = 0.0020. c,d, Average tumour volume (c) and survival fraction (d) of each group of\nMC38-bearing mice (n = 5 mice), two-tailed unpaired Student’s t-test, *p = 0.0274, ****p < 0.0001.\n5) The analysis time points are not aligned with Day 1/7 for B16F10 and Day 5 for MC38, which\nmay cause bias in the comparison. The difference in time points needs to be clearly explained.\n- We thank the reviewer for pointing out this important consideration. Firstly, the tumour volume\ngrowth rate is different in the two tumour models. Secondly, the response of the two tumours to\nBNCT is different. In view of this difference, the time points for analysis were selected based on the\ndifferential radiation sensitivity of the two tumour models (which is the inflection point of the tumour\ngrowth curve as shown). For MC38 tumours, as shown in Extended Data Fig. 1i, tumour volume\npeaked on Day 5 after BNCT treatment, which guided our selection of this time point for single-cell\nsequencing. In contrast, B16F10 tumours are less sensitive to BNCT, as indicated by both in vitro\nand in vivo experiments, so we extended the sampling to Day 7 to better capture treatment effects.\nAdditionally, to observe dynamic changes following irradiation, we also included samples collected\nat Day 1.\n6) There is an increase in the percentage of immune cells in the tumour after BNCT treatment, but\nthis may be a “relative increase” due to a decrease in tumour cells rather than an increase in the\nnumber of immune cells. Absolute count or density data should be presented.\n- We thank the reviewer for raising this important point. While we agree that relative percentages of\nimmune cells may be influenced by the decrease in tumour cells after BNCT, we consider immune\ncell proportions to be particularly relevant for understanding the immune landscape during tumour\nregression. A higher proportion of immune cells relative to tumour cells indicates the greater the\nimmune system's numerical advantage over tumour cells, which enhances the potential for tumour\nclearance. Even if the absolute density of immune cells is high, their ability to effectively eliminate\ntumour cells may be limited if the tumour cell population remains dominant.\nTo address the reviewer’s concern, we have quantified the absolute number of infiltrating immune\ncells. Compared with the control group, BNCT treatment significantly increased the counts of CD8+\nT cells, CD11c+ dendritic cells, and F4/80+ macrophages (Fig. R18). Furthermore, when compared\nwith X-ray treatment, BNCT also induced a significant increase in CD11c+ MHC-II+ DCs (Fig.\nR18c), which plays the important role in antigen presenting. These results indicated that BNCT not\nonly alters relative proportions but also increase the absolute infiltration of key immune cell\npopulations.\nFig. R18. a–e, Proportion of CD8+ T cells (a, *p = 0.0465, **p = 0.0072), CD11c+ myeloid cells (b, NC vs\nX-ray, ***p = 0.0010, NC vs BNCT, ***p = 0.0002), CD11c+MHC-II+ cells (c, NC vs X-ray, **p = 0.0058,\nNC vs BNCT, ***p = 0.0003, X-ray vs BNCT, ***p = 0.0007), F4/80+ macrophages (d, ***p = 0.0006,\n****p < 0.0001), and F4/80+MHC-II+ cells (e, NC vs X-ray, *p = 0.0204, NC vs BNCT, *p = 0.0198) in\ntumours. (n = 7 tumour sections, two-tailed unpaired Student’s t-test).\n7) ATP, HMGB1, and IFN have been evaluated, but ICD indicators such as surface calreticulin have\nnot been evaluated. Complementary discussions will provide a better understanding of ICD induction\nby BNCT.\n- We thank the reviewer for this suggestion. Following the reviewer’s suggestion, we have examined\ncalreticulin expression in tumours as shown in Fig. R19. The results indicated that BNCT treatment\ndoes not noticeably alter calreticulin expression. We speculated that the properties of α-particles and\nLi-7 particles may preferentially damage nuclear DNA rather than triggering the classic endoplasmic\nreticulum stress pathway that leads to calreticulin translocation. In addition, tumour cell type-specific\nresponses may also lead to a lack of calreticulin upregulation. We have added a discussion of these\nfindings in the revised manuscript to provide a more comprehensive view of ICD induction by BNCT.\nFig. R19. a, Representative immunofluorescence images of tumour samples. Nuclei were stained with DAPI\n(blue) and an antibody against Calreticulin (green). Scale bar, 50 μm. b, Quantification of relative Calreticulin\ngreen fluorescence per cell. n = 10 tumour sections.\n8) While NK cell activity has been suggested by scRNA-seq, functional verification (e.g., NK cell\nremoval experiments) has not been performed. It would be more comprehensive if added to the\ndiscussion as a future issue.\n- We thank the reviewer for this valuable suggestion. Following the reviewer’s advice, we performed\nNK cell depletion experiments using anti-NK antibodies in mice. We found that the absence of NK\ncells did not affect the therapeutic efficacy of BNCT (Fig. R20), indicating that although NK cells\ncould be activated after BNCT, they were not essential for the anti-tumour immune effects. We have\nadded these data and discussion in the revised manuscript to clarify the role of NK cells.\nFig. R20. Average tumour volumes (a,b) and survival fractions (c) of each group of MC38-bearing mice\ntreated with BNCT or the combination of BNCT and an anti-NK antibody (n = 5 mice).\n9) Single-cell RNA-seq and TCR data are not publicly available and insufficient in terms of\nreproducibility; registration with GEO and ArrayExpress and publication of analysis codes on GitHub\nare desired.\n- We thank the reviewer for highlighting this important point regarding data availability. During the\ninitial anonymous review, the single-cell RNA-seq and TCR data were not publicly released to\nmaintain confidentiality. We have now uploaded the data to the Genome Sequence Archive (GSA)\ndatabase, and the data will be made fully publicly available upon acceptance of the manuscript. In\naddition, all analysis codes have been uploaded to GitHub, ensuring reproducibility of the results. We\nwill include this information in the revised manuscript to facilitate access and transparency.\nGSA database temporary access: https://ngdc.cncb.ac.cn/gsa/s/8Ye82V1v\nGitHub: https://github.com/Qi-1111/BNCT_scripts\nMinor Issues:\n1) There are several typos, including “gliomas” for “gliomass” in line 76, “themice” for “the mice”\nin line 590, and “filte” in line 595. They need to be corrected.\n2) Figure reference formatting: In one instance, a duplicate reference is present: “Extended Data Fig.\nFig. 2a,b” has “Fig.” written twice. This appears to be a minor editorial slip. (Line 121)\n- We thank the reviewer for pointing out these typographical and formatting errors. We have carefully\ncorrected all typos, including “gliomass”→“gliomas”, “themice”→“the mice”, and “filte”\n→ “filter”. In addition, the duplicate figure reference (“Extended Data Fig. Fig. 2a,b”) has been\ncorrected. We appreciate the reviewer’s careful reading and attention to these details."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed all my concerns, and I support the publication of the manuscript.\nIf the other reviewers and the editor have deemed the paper acceptable, I think it should be accepted.", "reviewer_2": "The authors have incorporated comparisons between X-ray and BNCT in Figure 2 and Extended Figure 3, as requested.\nTo enhance clarity for the reader, it would be beneficial to include the radiation dose (in Gy) used in the experiments shown in Figure 2 (as well as in other figures) directly within the figure legend, in addition to the Methods section. This would allow readers to immediately identify a key parameter of the experimental setup.\nPlease note that the legend for the B16F10 condition is missing in panel B of Figure 2.\nThe authors have also added several experiments to support the induction of CICD by BNCT. These additions strengthen the manuscript. Although some aspects regarding the relevance of CICD in this context remain to be fully elucidated, the authors have addressed this limitation in the Discussion section.\nThe functional assays demonstrating that DC activation enhances T cell responses, but not NK cell responses, are also valuable and provide further mechanistic insight.\nOverall, the manuscript has been significantly improved and is suitable for publication.", "reviewer_4": "This assessment has been made based on a limited-time review of the revised manuscript, and it is possible that some points noted below are already described elsewhere in the text. I hope these comments are of some assistance.\nThe accelerator-based neutron generator used in this study appears to be highly novel. Although previous reports are cited, the neutron beam characteristics (such as spectrum, irradiation geometry, and dose rate) are not clearly described. To help readers understand the distinct properties of this system and to facilitate comparison with other neutron sources, I recommend adding a concise explanation or summary of the beam quality parameters."}, "author_response": "Responses to reviewers’ comments:\nDear reviewers,\nOn behalf of the authorship group, I would like to thank you for taking the time to evaluate our\nmanuscript. We are deeply grateful for your appreciation of our work. Please kindly find our point-\nby-point response to the reviewers’ comments below.\nReviewer #1 (Remarks to the Author):\nThe authors have adequately addressed all my concerns, and I support the publication of the\nmanuscript. If the other reviewers and the editor have deemed the paper acceptable, I think it should\nbe accepted.\n- We thank the reviewer for the comment on our work.\nReviewer #2 (Remarks to the Author):\nThe authors have incorporated comparisons between X-ray and BNCT in Figure 2 and Extended\nFigure 3, as requested. To enhance clarity for the reader, it would be beneficial to include the radiation\ndose (in Gy) used in the experiments shown in Figure 2 (as well as in other figures) directly within\nthe figure legend, in addition to the Methods section. This would allow readers to immediately\nidentify a key parameter of the experimental setup.\n- We thank the reviewer for the suggestion, the radiation dose has been added in the figure legend.\nPlease note that the legend for the B16F10 condition is missing in panel B of Figure 2.\n- We thank the reviewer for pointing this out, we have now added the missing label and updated the\nfigure.\nThe authors have also added several experiments to support the induction of CICD by BNCT. These\nadditions strengthen the manuscript. Although some aspects regarding the relevance of CICD in this\ncontext remain to be fully elucidated, the authors have addressed this limitation in the Discussion\nsection. The functional assays demonstrating that DC activation enhances T cell responses, but not\nNK cell responses, are also valuable and provide further mechanistic insight. Overall, the manuscript\nhas been significantly improved and is suitable for publication.\n- We sincerely thank the reviewer for the positive evaluation and encouraging comments.\nReviewer #4 (Remarks to the Author):\nThis assessment has been made based on a limited-time review of the revised manuscript, and it is\npossible that some points noted below are already described elsewhere in the text. I hope these\ncomments are of some assistance. The accelerator-based neutron generator used in this study appears\nto be highly novel. Although previous reports are cited, the neutron beam characteristics (such as\nspectrum, irradiation geometry, and dose rate) are not clearly described. To help readers understand\nthe distinct properties of this system and to facilitate comparison with other neutron sources, I\nrecommend adding a concise explanation or summary of the beam quality parameters.\n- We thank the reviewer for the helpful suggestion. The relevant neutron beam characteristics have\nbeen added to the Methods section."}]

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