Unleashing the potential of bimetallic nanobomb-mediated STING pathway to enhance bispecific T-cell engager against colorectal cancer photo-immunotherapy

Mu, Min; Li, Hui; Chen, Bo; Fan, Rangrang; Zhong, Kunhong; Wang, Guoqing; Feng, Chenqian; Chen, Nianyong; Guo, Gang; Tong, Aiping; Zou, Bingwen

doi:10.1038/s41392-026-02596-6

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Published: 05 March 2026

Unleashing the potential of bimetallic nanobomb-mediated STING pathway to enhance bispecific T-cell engager against colorectal cancer photo-immunotherapy

Signal Transduction and Targeted Therapy volume 11, Article number: 80 (2026) Cite this article

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Abstract

The broader clinical application of Bispecific T-cell engagers (BiTEs) is hindered by their short half-life, on-target off-tumor toxicity, and limited therapeutic effect for solid tumors. Herein, we constructed a bimetallic-enriched triple-kill nanobomb manganese/Co²⁺-dopamine@BiTE/HPT (MnO₂/Co-DA@BiTE/HPT) based on metal-polyphenol to improve the immunosuppressive tumor microenvironment by activating innate and adaptive immunity, thereby enhancing the treatment efficacy of BiTEs (PD-L1/CD3). A hyaluronic acid-modified PD-L1 aptamer (HPT) was introduced to improve the active targeting of the nanobombs and bind with PD-L1 overexpressing colorectal cancer. Bimetallic (Mn²⁺/Co²⁺) activated the STING pathway; simultaneously, photothermal therapy (PTT) induces DNA fragmentation to cooperate with bimetallic to amplify the STING signal to “heat” the “cold” tumor microenvironment. The “hot” tumor with a large amount of T-cell infiltration facilitated BiTE recruitment of T cells to kill tumor cells. Furthermore, the efficient therapeutic potency of the triple-kill nanobombs (STING, BiTE, and PTT) was determined in subcutaneous colorectal cancer, distal, lung metastasis, and postoperative recurrence models, which indicated that MnO₂/Co-DA@BiTE/HPT could improve the immune microenvironment, produce long-term immune memory, inhibit tumor growth, and prevent tumor recurrence and metastasis.

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Introduction

Immunotherapy, the fourth type of therapy after surgery, chemotherapy, and radiotherapy, has made revolutionary progress in cancer treatment by specifically activating the immune system against tumors.^1,2 Bispecific T-cell engagers (BiTEs) are unique-targeted antibodies that recruit cytotoxic T cells against tumors by simultaneously targeting CD3 and tumor-specific antigens to promote selective T-cell killing responses.^3,4 BiTE, consisting of bi-antigen specificity, can bind to two unique antigens at the same time, thus facilitating cell-to-cell interactions, which is different from antibodies that have a single antigen specificity.⁵ Blinatumomab, a CD3/CD19-specific antibody, was approved by the Food and Drug Administration (FDA) in 2014 for the treatment of acute lymphoblastic leukemia.⁶ More than 50 CD3-based T-cell BiTE antibodies, including Her2/CD3 and PSMA/CD3, are currently in clinical trials.⁷ However, the therapeutic effect of BiTE appears to be limited to immunogenic tumors that exhibit substantial lymphocyte infiltration and tumor-associated antigen (TAA) expression.^8,9 Additionally, the clinical application of BiTE therapy in solid tumors has been limited by challenges such as short half-life, “off-target” effects, and immune tolerance. To address these issues, various nanomaterial-based delivery systems have been developed to encapsulate BiTEs, enhancing their stability, targeting capability, and safety profile. These include SiTE, PSTAGylated BiTE, S-biAb/dEGCG@NPs, and BiTEs-GSP-NCs.^10,11,12,13 For instance, Gong et al. developed a supramolecular bispecific T cell engager via the infusion of the small-molecule drug amantadine, which can control the anti-tumor activity and off-tumor toxicity of bispecific antibodies.¹⁰ Fan et al. used S-biAb/dEGCG@NPs nanoparticles as a delivery vehicle for B7-H3×CD3 BiTEs to improve therapeutic outcomes in glioblastoma therapy.¹² Despite these promising advances, research on efficient delivery strategies for BiTEs remains relatively limited.

STING (stimulator of interferon genes), a signal transduction molecule involved in the virus-induced innate immune response, is currently a hot target for drug research and development.^14,15 STING has rapidly expanded the field of research on immunology and tumor immunotherapy.¹⁶ The cGAS-STING pathway can sense not only exogenous danger signals from viruses and bacteria, but also endogenous danger signals from damaged mitochondria and endocytosis fragments of dead tumor cells.^17,18 Activation of the STING pathway in antigen-presenting cells (APCs) may produce IFN-I to recruit and cross-prime effector T cells to overcome the “cold” tumor barrier.^19,20,21 An increasing number of studies have begun to investigate the activation of the innate immune system by Mn²⁺.^22,23,24 Mn²⁺, as the second cyclic GMP-AMP synthase (cGAS) activator in addition to DNA, can directly activate cGAS, which can enhance the body’s immune surveillance and immune clearance of tumors.²² The nano-STING combination with chemotherapy, radiotherapy, chimeric antigen receptor T-cell (CAR-T) therapy, checkpoint inhibitors, and other novel therapeutic methods can synergistically enhance the antitumor effect.^25,26

Herein, bimetallic nanobombs manganese/Co²⁺-dopamine (MnO₂/Co-DA) with dual functions of photothermal therapy (PTT) and STING activation were formed by coordination of dopamine and Co²⁺ on MnO₂ (Scheme 1). To enhance the ability of MnO₂/Co-DA nanobombs to regulate the tumor microenvironment and overcome the off-target effects of PD-L1/CD3 BiTE, as well as the weak response of BiTE in “cold” tumors, BiTE was successfully loaded with MnO₂/Co-DA by combining Co²⁺ with His tags in BiTE to realize the combination of the STING pathway and BiTE. Hyaluronic acid (HA)-modified PD-L1 aptamer (HPT) was introduced to prevent off-target effects and improve the active targeting of drugs. Under the self-assembly of HPT, the hierarchical loading of BiTE was realized, obtaining MnO₂/Co-DA@BiTE/HPT nanobombs. The use of PTT induced DNA fragmentation to cooperate with Mn²⁺ and Co²⁺ to amplify the signal of the STING pathway, activating the innate immune system. Faced with hyaluronidase-rich tumor tissue, MnO₂/Co-DA@BiTE/HPT nanobombs could rapidly depolymerize and release drugs to activate the innate and adaptive immune systems, reverse the tumor immunosuppressive microenvironment, and achieve multitarget tumor therapy.

Results

Screening of metal ions

First, we successfully extracted and cultured bone marrow-derived dendritic cells (BMDCs). To investigate whether metal ions other than Mn²⁺ could specifically induce the secretion of IFN-β and the maturation of BMDCs. Several metal ions (Na⁺, Ca²⁺, Fe³⁺, Co²⁺) were added to incubate with BMDCs. The enzyme-linked immunosorbnent assay (ELISA) results showed that the level of interferon-β (IFN-β) was enhanced after treatment with Mn²⁺ and Co²⁺ (Fig. 1a). The phosphorylated TANK binding kinase 1 (pTBK1), phosphorylated interferon regulatory factor 3 (pIRF3), and phosphorylated STING (pSTING) proteins were analyzed to further evaluate the effects of Mn²⁺ and Co²⁺ on the STING pathway (Fig. 1b and Supplementary Fig. 1). The results showed that the addition of Mn²⁺ and Co²⁺ increased the expression levels of several phosphorylation proteins, which was consistent with a previous study. Subsequently, to evaluate whether the metal ions synergistically activate the STING pathway with MnO₂, MnO₂/Na-DA, MnO₂/Ca-DA, MnO₂/Ce-DA, and MnO₂/Co-DA nanosolutions were applied to simulate BMDCs. The ELISA (Supplementary Fig. 2a) and western blot (WB) (Supplementary Figs. 2b and 3) results both indicated that MnO₂/Co-DA could amplify the activation of the STING pathway owing to the synergy of Co²⁺ and MnO₂. Therefore, the MnO₂/Co-DA solution was selected as the STING activator for the subsequent experiments.

Characterization of BiTE

BiTE can bind CD3 and TAA, leading to the formation of immune synapses between T cells and tumor cells (Fig. 1c). The PD-L1/CD3 BiTE antibody with His tag was expressed by HEK293T cells (Supplementary Fig. 4). Flow cytometry was used to detect the binding affinity of PD-L1 to CT26 cells (high expression of PD-L1) and CD3 to CD8⁺ T cells (high expression of CD3) in BiTE structures (Supplementary Fig. 5). As shown in Fig. 1d, PD-L1/CD3 BiTE could specifically bind CT26 cells and CD8⁺ T cells. Compared with IgG, BiTE had a stronger binding affinity with CT26 and CD8⁺ T cells with increasing concentration. When T cells/CT26 cells were cultured with BiTE, the level of IFN-γ (Supplementary Fig. 6a) and the proportion of activated T cells (Supplementary Fig. 6b) increased with increasing BiTE concentration. Additionally, Supplementary Fig. 7 shows that T cells surrounded CT26 cells and lysed tumor cells after treatment with BiTE. The killing effect of T cells became more obvious with increasing BiTE concentration. However, fewer T cells attacked tumor cells without BiTE treatment, which showed that BiTE was effective in recruiting T cells to attack tumor cells.

Characterization of the MnO₂/Co-DA@BiTE/HPT nanobombs

The above results inspired us to design a strategy to combine STING activators and BiTE with immune-activating therapies. First, HA-PD-L1(HPT) was synthesized by the reaction of the carboxyl group on HA and the amino-modified PD-L1 aptamer (Supplementary Fig. 8a). To verify the successful synthesis of HPT, the tetramethylrhodamine (TAM)-labeled PD-L1 aptamer was used to react with HA, and TAM-HPT was detected by agarose electrophoresis. As shown in Supplementary Fig. 8b, compared with the PD-L1 aptamer, the position of the band of HPT and TAM-HPT moved upwards, indicating that HPT was successfully synthesized.²⁷

MnO₂ nanoparticles were prepared by the potassium permanganate (KMnO₄) reduction method.²⁸ The MnO₂/Co-DA nanobombs were formed by the coordination of Co²⁺ and DA. Due to the Co²⁺ in MnO₂/Co-DA, BiTE could be successfully captured on the surface of MnO₂/Co-DA by combining with the His tag of BiTE.²⁹ Additionally, previous studies have reported the ability of DA to capture antibodies.^30,31 Therefore, MnO₂/Co-DA@BiTE nanobombs were prepared through the interaction between Co²⁺/DA and BiTE. Finally, MnO₂/Co-DA@BiTE/HPT nanobombs were obtained via the self-assembly of HPT. The introduction of HPT could enhance the targeting ability of the nanobombs.³² The MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT nanobombs had particle sizes of ~85.7 nm and 167.2 nm, respectively (Fig. 1e and Supplementary Fig. 9). To confirm the loading of BiTE in the MnO₂/Co-DA@BiTE/HPT nanobombs, we performed circular dichroism (CD) analysis using free PD-L1/CD3 BiTE as the standard, and found that BiTE was present in the MnO₂/Co-DA@BiTE/HPT nanobombs (Fig. 1f). The results of molecular docking showed that DA of Co-DA structure could form hydrophobic and hydrogen bonding with amino acids of BiTE (Fig. 1g). Additionally, Co²⁺ of Co-DA structure formed a coordination effect with the His tags of BiTE (Fig. 1h). These results showed that BiTE molecules could bind to the Co-DA structure of MnO₂/Co-DA. Supplementary Fig. 10 shows that MnO₂/Co-DA@BiTE/HPT nanobombs were unstable in the PBS buffer. And MnO₂/Co-DA@BiTE/HPT nanobombs displayed stability during 36 h in water and saline. The percentage of BiTE released from the MnO₂/Co-DA@BiTE/HPT nanobomb was tested in PBS with different concentrations of HAase, and a high release rate was observed with increasing hyaluronidase (HAase) concentration (Supplementary Fig. 11).

The ultraviolet-visible spectroscopy (UV‒Vis) spectrum showed that MnO₂/Co-DA nanobombs had absorption peaks in the range of 600–800 nm (Supplementary Fig. 12), which should contribute to the formation of a network structure between metals and phenols, forming an absorption peak in the near infrared.^33,34 In addition, the X-ray photoelectron spectroscopy (XPS) results of MnO₂/Co-DA@BiTE/HPT indicated the presence of Mn and Co (Supplementary Fig. 13). Two characteristic peaks at 643 eV (Mn2p3/2) and 654.39 eV (Mn2p1/2) were attributed to MnO₂ (Supplementary Fig. 14). The characteristic peaks at 782.69 eV (Co2p3/2) and 798.19 eV (Co2p1/2) were attributed to Co. Inductively coupled plasma-mass spectrometry (ICP‒MS) showed that the contents of Mn and Co were 20.83% and 14.33%, respectively. Supplementary Fig. 15 indicates that the peaks at 24°, 37°, and 67° were attributed to MnO₂.³⁵ Moreover, there was no obvious spectrum peak of MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT, indicating that the MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT nanobombs were amorphous. The MnO₂/Co-DA nanobombs were irradiated by an 808 nm laser to evaluate the photothermal conversion effect. As shown in Supplementary Fig. 16, the temperature of the MnO₂/Co-DA nanobombs increased gradually with increasing irradiation time. Additionally, the temperature variation of MnO₂/Co-DA was positively correlated with laser power and concentration (Supplementary Fig. 17). The photothermal conversion efficiency of MnO₂/Co-DA was 30.23% (Supplementary Fig. 18), and the nanobombs had great photothermal stability (Supplementary Fig. 19).

Methylene blue (MB) was used as an indicator to detect hydroxyl radical (•OH). As shown in Supplementary Fig. 20, there was no significant MB degradation in the MB and MB+H₂O₂ groups. In contrast, after the addition of MnO₂/Co-DA, the spectrum of MB was weakened, and the intensity of the absorption spectrum became weaker with increasing incubation time (Supplementary Fig. 21). It is speculated that a Fenton-like reaction occurred between Co²⁺ and Mn²⁺. MnO₂ reacted with H₂O₂ to form Mn²⁺ and O₂. The bubbles are O₂ (Supplementary Fig. 22a), which is produced at a faster rate as the acidity decreases. Supplementary Fig. 22b shows that the UV‒Vis spectrum intensity of MnO₂ became weaker with increasing time, indicating the degradation of MnO₂.

The function of BiTE in MnO₂/Co-DA@BiTE/HPT in vitro

First, the expression of PD-L1 on the surface of CT26 cells was detected by immunofluorescence. As illustrated in Supplementary Fig. 23, the PD-L1 receptor was highly expressed on the surface of CT26 cells. BiTE has biological activity, which could be affected by external conditions. To evaluate the influence of BiTE binding with MnO₂/Co-DA on protein biological activity, MnO₂/Co-DA@BiTE/HPT nanobombs were directly treated with T/CT26 cells. When the BiTE concentration was 25 nM, the proportions of CD25⁺CD69⁺ cells in the BiTE and MnO₂/Co-DA@BiTE/HPT groups were 23.54% and 21.52%, respectively (Supplementary Fig. 24a). The proportion of CD25⁺CD69⁺ cells increased with increasing concentrations of BiTE (Supplementary Fig. 24b). Furthermore, the BiTE and MnO₂/Co-DA@BiTE/HPT groups exhibited increased expression of IFN-γ (Supplementary Fig. 24c). The above results indicated that the biological function of BiTE was preserved in MnO₂/Co-DA@BiTE/HPT nanobombs. Additionally, the affinity of BiTE was detected by a PD-L1 ELISA kit after irradiation with an 808 nm laser. Compared with the nonlaser treatment group, ~85.09% of BiTE activity was retained after irradiation with a 0.75 W cm⁻² 808 nm laser (Supplementary Fig. 25), indicating that the effect of PTT on the biological activity of BiTE was negligible.

Cell cytotoxicity assay

MnO₂/Co-DA@BiTE/HPT could trigger the release of damage-associated molecular patterns (DAMPs), tumor-derived dsDNA, and metal ions (Mn²⁺ and Co²⁺) with irradiation with an 808 nm laser, which could induce the maturation of DCs and inhibit tumors (Fig. 2a). Figure 2b shows that the cytotoxicity of MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT was concentration dependent. The activity of CT26 cells was further reduced by 808 nm laser irradiation. In addition, the RTCA system was applied to monitor the toxicity of different concentrations of MnO₂/Co-DA@BiTE/HPT (Supplementary Fig. 26). Cell apoptosis was used to further analyze cytotoxicity (Supplementary Fig. 27). The BiTE group did not induce tumor cell apoptosis. MnO₂ can be rapidly degraded to produce Mn²⁺ under high levels of H₂O₂ in tumor cells.³⁶ Bimetallic Mn²⁺ and Co²⁺ could generate reactive oxygen species (ROS) via a Fenton-like reaction to induce tumor cell apoptosis. Thus, cell apoptosis was detected in the MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT groups. The apoptosis-inducing ability of the MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT groups was improved after laser irradiation. The cell cytotoxicity of MnO₂/Co-DA@BiTE/HPT was superior to that of MnO₂/Co-DA nanobombs. The reason is that the targeting of HPT promotes more MnO₂/Co-DA@BiTE/HPT to bind to tumor cells, thus exerting a strong antitumour effect. To more directly observe the activity of MnO₂/Co-DA@BiTE/HPT against CT26 cells, a live and dead kit was used to stain the cells. Supplementary Fig. 28 shows that the NS and BiTE groups displayed strong green fluorescence signals. In contrast, both green and red signals were present in the MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT groups. After laser irradiation, the red signal was significantly enhanced in the two groups, revealing that the combination of Fenton toxicity and hyperthermia amplified the tumor-killing power.

ROS play an important role in tumor therapy, which can not only directly kill tumors, but also induce tumor cells to produce immunogenic cell death (ICD) in coordination with immunotherapy.^37,38 The efficiency of the Fenton reaction was detected by an ROS probe in CT26 cells. As shown in Supplementary Fig. 29, Mn²⁺ and Co²⁺ could induce the production of •OH via a Fenton-like reaction. Therefore, the MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT groups emitted green fluorescence signals. The intensity of green signals was increased under laser irradiation, which contributed to the fact that PTT promoted rapid depolymerization of the nanobombs and released more metal ions, thus producing more •OH.

dsDNA generation and ICD induction

Excessive oxidative stress in cells can lead to the destruction of cell homeostasis, and the decline in mitochondrial membrane potential is a sign of early apoptosis of tumor cells.³⁹ JC-1 is an ideal fluorescent dye for detecting changes in mitochondrial membrane potential. As illustrated in Supplementary Fig. 30, strong red fluorescence signals were observed in the control and BiTE groups, indicating that BiTE did not affect mitochondrial function. The fluorescence signals were weakened in the MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT groups. The red fluorescence signal disappeared under the synergistic action of PTT and metal ions. Mitochondrial stress, such as virus-induced mitochondrial stress, leads to leakage of mitochondrial DNA (mtDNA) into the cytoplasm, activating the STING pathway and promoting the production of cytokines.⁴⁰ Cell apoptosis induces mitochondrial stress, and then leaks mtDNA, thus assisting Mn²⁺ in activating the STING pathway. In addition, ROS will damage DNA in mitochondria and the nucleus.⁴¹ Therefore, damaged dsDNA was detected by γ-H2Aχ immunofluorescence in CT26 cells after treatment with different formulations. γ-H2Aχ is a marker of double-stranded DNA (dsDNA) breakage and an important tool for studying DNA damage. γ-H2Aχ-positive staining was observed in the MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT groups in Fig. 2c, especially after laser treatment. These results showed that MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT could break dsDNA in CT26 cells with the aid of PTT. Previous studies have shown that PTT can improve antitumor immunity by inducing ICD in tumor cells, expressing calreticulin (CRT), and releasing high mobility group box 1 (HMGB1) and adenosine 5′-triphosphate (ATP). ICD acts as a bridge between tumor cells and the microenvironment by enhancing the immunogenicity of tumor cells. CRT can be transported to the surface of dying tumor cells to promote TAA uptake by APCs. A weak red fluorescence signal of CRT was observed, as well as a reduced HMGB1 signal in MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT groups. After laser treatment, the intensity of ICD was significantly increased (Fig. 2d).

Activation of the STING pathway

Mn²⁺ and Co²⁺ can activate the STING pathway of DCs to release IFN-β, thus inducing the maturation of DCs.^25,42 Subsequently, different concentrations of MnO₂/Co-DA nanobombs were introduced to investigate the maturation ability of BMDCs. As shown in Supplementary Fig. 31a, the proportion of mature BMDCs increased with increasing MnO₂/Co-DA concentration. The level of IFN-β in the cell supernatant was also increased (Supplementary Fig. 31b), which further indicated that MnO₂/Co-DA could induce the maturation of BMDCs.

A transwell system was used to study the immune response induced by MnO₂/Co-DA@BiTE/HPT nanobombs in vitro (Supplementary Fig. 32). The results showed that the expression level of CD80⁺CD86⁺ on BMDCs was 32.76% in the MnO₂/Co-DA@BiTE/HPT+L group, which was higher than that of the MnO₂/Co-DA@BiTE/HPT group (22.39%) (Fig. 2e). MnO₂/Co-DA@BiTE/HPT nanobombs could generate ROS to produce cytotoxicity, promote tumor cell death, and release TAAs. After laser irradiation, CT26 cells could release a large amount of TAAs (tumor cell debris, damaged dsDNA, etc.), which could significantly promote the maturation of BMDCs to secrete IFN-β (Fig. 2f). The expression of STING pathway-related proteins in DC2.4 and RAW264.7 cells after treatment with nanobombs. Figure 2g and Supplementary Fig. 33 show that the levels of p-TBK1, p-STING, and p-IRF3 were increased in the MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT groups. Especially after laser treatment, PTT promoted tumor cells to generate ICD, and high temperature caused the death of CT26 cells to release dsDNA, which promoted APCs to phagocytose dsDNA to cooperate with Mn²⁺ and Co²⁺ to enhance the levels of p-TBK1, p-STING, and p-IRF3.

Antitumour assay in the simulated immune system

DCs are professional APCs that process antigens to combine with major histocompatibility complex (MHC) molecules to form a complex and bind with T-cell receptors, thus triggering the whole process of T-cell activation.⁴³ The costimulatory molecules CD80 and CD86 on DCs bind to CD28 on the T-cell surface, thus activating T lymphocytes, stimulating the activation and proliferation of initial T cells, and initiating the immune response. Figure 2f shows that MnO₂/Co-DA@BiTE/HPT could induce the maturation of BMDCs. Therefore, we investigated whether MnO₂/Co-DA@BiTE/HPT promotes T-cell activation in vitro (Supplementary Fig. 34). As shown in Fig. 2h, the percentage of initial T cells activated into CD8⁺ T cells by BiTE was 15.20% in the T-cell/BMDC/CT26 coculture system. BiTE designed for PD-L1 could enhance T-cell function by blocking the interaction between PD-L1/PD-1. Therefore, PD-L1/CD3 BiTE could stimulate the proliferation and activation of initial T cells into CD8⁺ T cells. When the initial T cells were treated with MnO₂/Co-DA@BiTE/HPT, the proportion of CD8⁺ T cells was 19.30%, which was higher than that of the BiTE and MnO₂/Co-DA groups (Fig. 2i). PTT induced the death of CT26 cells to release dsDNA, which could promote the maturation of BMDCs. Mature BMDCs process TAAs released by CT26 cells and bind to T cell receptor (TCR) to further stimulate T-cell activation and proliferation. Therefore, PTT could increase the proportion of activated CD8⁺ T cells. Additionally, compared with NS, IFN-γ levels after treatment were increased. The level of IFN-γ in MnO₂/Co-DA@BiTE/HPT+L group was significantly higher than that in the other groups (Fig. 2j).

A T-cell/BMDC/CT26 coculture system was constructed to investigate the antitumour effect of the immune system in vitro (Supplementary Fig. 35). After treatment with MnO₂/Co-DA@BiTE/HPT+L, the viability of CT26 cells was 4.39%, showing a significant antitumour effect. The results demonstrated that the combination of the STING pathway, BiTE therapy, and PTT had a synergistic effect on the killing effect of tumor cells.

Pharmacokinetics and biodistribution of MnO₂/Co-DA@BiTE/HPT in vivo

To obtain the pharmacokinetics of MnO₂/Co-DA@BiTE/HPT in blood, CT26 tumor-bearing mice were intravenously injected with MnO₂/Co-DA@BiTE/HPT. As shown in Supplementary Fig. 36, the blood circulation half-life was detected to be 4.18 h. The distribution of MnO₂/Co-DA@BiTE/HPT in CT26 tumor-bearing mice was determined after systemic administration. The infrared dye Cy5.5 was used to label the nanobombs to reduce the interference of mouse fluorescence and facilitate observation. Cy5.5/MnO₂/Co-DA nanobombs could effectively aggregate in tumor tissues, and the fluorescence signal could still be detected at 96 h, meaning that the nanobombs could accumulate in tumor tissues for a long time (Supplementary Fig. 37). In addition, after the introduction of HPT, the fluorescence intensity of MnO₂/Co-DA@BiTE/HPT was significantly higher than that of the MnO₂/Co-DA group. The reason is that the PD-L1 aptamer could enhance the specific targeting ability of the MnO₂/Co-DA@BiTE/HPT nanobombs. To further confirm that the PD-L1 aptamer can specifically enhance tumor targeting, we selected a nucleic acid molecule with a similar molecular weight to the PD-L1 aptamer but without any targeting function as a control (MnO₂/Co-DA@BiTE/HR). The results showed that the enrichment level of MnO₂/Co-DA@BiTE/HR was lower than that of MnO₂/Co-DA@BiTE/HPT group. Additionally, the major organs and the tumor tissues were collected at 96 h for ex vivo analysis (Supplementary Fig. 38). Strong fluorescence signals were observed in tumors. The fluorescence signal of MnO₂/Co-DA@BiTE/HPT was higher than that of MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HR groups. Aptamers, also known as “chemical antibodies”, can selectively bind to specific targets. Therefore, PD-L1 aptamers could target colorectal cancer with high expression of PD-L1.

In vivo antitumour efficacy of MnO₂/Co-DA@BiTE/HPT

To investigate whether MnO₂/Co-DA@BiTE/HPT nanobombs have significant antitumour effects. The subcutaneous tumor model of CT26 was established (Fig. 3a). The tumor-bearing mice were divided into six groups to be treated with different formulations. The temperature of the MnO₂/Co-DA group rose to 40 °C, while the tumor temperature was 52.3 °C in MnO₂/Co-DA@BiTE/HPT group (Supplementary Fig. 39). As shown in Fig. 3b and Supplementary Fig. 40, CT26-bearing mice treated with BiTE and MnO₂/Co-DA displayed a slight inhibition of tumor growth. However, the mice receiving MnO₂/Co-DA@BiTE/HPT+L treatment presented stronger tumor growth inhibition owing to the effective activation of the adaptive and innate immune systems. On the 18th day, the tumors were collected, weighed, and photographed (Fig. 3c). A similar antitumour trend was observed after treatment with different formulations in Fig. 3d. The weight of tumor-bearing mice did not significantly change after treatment with different formulations (Fig. 3e). Subsequently, H&E, Ki67, and TUNEL staining were carried out (Fig. 3f). Mice treated with MnO₂/Co-DA@BiTE/HPT+L showed the most marked tumor necrosis, proliferation inhibition, and cell apoptosis.

To explore the immune response after treatment with different formulations, the tumors were collected to analyze the immune cells by flow cytometry (Supplementary Fig. 41). CD8⁺ T cells in MnO₂/Co-DA@BiTE/HPT+L tumors were threefold higher than those in NS mice (Fig. 3g). Similarly, the immunofluorescence staining images further indicated that MnO₂/Co-DA@BiTE/HPT+L improved more tumor infiltration of CD4⁺ and CD8⁺ T cells compared with the other groups (Fig. 3h). Moreover, the mice treated with MnO₂/Co-DA@BiTE/HPT+L exhibited enhanced expression of IFN-γ. Treg cells in the tumor microenvironment could promote the immune escape of tumor cells. Therefore, we analyzed Treg cells after treatment with different formulations. As shown in Fig. 3i and Supplementary Fig. 42, the proportion of Treg cells decreased after treatment. Compared with that in the NS group, the proportion of Treg cells decreased to 9.52% in the MnO₂/Co-DA@BiTE/HPT+L group. The maturation of DCs infiltrated in tumors has important significance for the strength of the T-cell immune response and the prognosis of tumor patients. Activation of the STING pathway improves the maturation of DCs. Thus, the DCs in tumors were analyzed. MnO₂/Co-DA@BiTE/HPT increased the frequency of mature DCs to 38.41%, compared with 17.17% in the NS group. The DC maturation ratio of the MnO₂/Co-DA@BiTE/HPT group was markedly increased to 50.10% upon laser irradiation (Fig. 3j). Additionally, to assess the systemic immune response, we further examined the proportion of mature DCs in tumor-draining lymph nodes and Tems in the spleen. MnO₂/Co-DA@BiTE/HPT+L effectively enhanced DC maturation in the tumor-draining lymph nodes of mice (Supplementary Fig. 43 and Fig. 3k). The proportion of Tem increased from 10.11% to 33.51% (Supplementary Fig. 44 and Fig. 3l). Moreover, the mice treated with MnO₂/Co-DA@BiTE/HPT+L showed improved levels of the cytokines IFN-β, TNF-α, IL-6, and IFN-γ (Fig. 3m). The results indicated that the MnO₂/Co-DA@BiTE/HPT nanobombs could effectively stimulate the systemic immune response for immunotherapy.

Subsequently, we explored the biological mechanisms of MnO₂/Co-DA@BiTE/HPT+L on tumors by RNA sequencing. The Venn diagram suggested that there were significant discrepancies among primary transcripts between the NS and MnO₂/Co-DA@BiTE/HPT+L groups (Fig. 4a). Compared with the NS group, 866 upregulated genes and 525 downregulated genes were observed in the tumors treated with MnO₂/Co-DA@BiTE/HPT+L (Fig. 4b). To further understand the potential targeting pathway of MnO₂/Co-DA@BiTE/HPT+L, KEGG analysis showed that some cGAS-STING related pathways were significantly upregulated compared with those in NS mice, including chemokine signaling, the IL-17 signaling pathway, cytokine and cytokine receptor interaction, TNF signaling, and the cytoplasmic DNA sensing pathway, which could induce a systemic immune response (Fig. 4c). Additionally, Fig. 4d shows that most pathways in tumors were upregulated after MnO₂/Co-DA@BiTE/HPT+L treatment because the number of upregulated enriched genes in each pathway was higher than that of downregulated genes. Studies have shown that Mn²⁺ can enhance IFN-I, proinflammatory cytokines, and chemokines by activating the cGAS-STING pathway. Co²⁺ and dsDNA could synergistically amplify the activation of the STING pathway. Subsequently, gene set enrichment analysis (GSEA) was applied to determine the impact of MnO₂/Co-DA@BiTE/HPT+L on the corresponding signaling pathways at immunotherapy (Fig. 4e). The results showed positive enrichment of inflammatory, TNFA, and IFN-γ in MnO₂/Co-DA@BiTE/HPT+L-treated tumors (Fig. 4f–h).

Additionally, to elucidate the antitumour mechanism of MnO₂/Co-DA@BiTE/HPT+L, we first studied the RNA and protein levels of the STING pathway in tumor tissues. The expression levels of IFNa4, IFIt1, IFNb, Isg15, and IFN-β were significantly increased (Fig. 4i). Moreover, WB analysis revealed that the protein levels of p-IRF3 and p-STING were robustly upregulated in MnO₂/Co-DA@BiTE/HPT+L mice (Fig. 4j and Supplementary Fig. 45).

MnO₂/Co-DA@BiTE/HPT boosted antitumour immunotherapy

A CT26 bilateral tumor model was used to evaluate the antitumour effect of MnO₂/Co-DA@BiTE/HPT (Fig. 5a). As shown in Fig. 5b, MnO₂/Co-DA@BiTE/HPT, synergizing with PTT, displayed an obvious inhibitory effect on distal tumors. On the 12th day, the tumors were collected, weighed, and photographed (Fig. 5c). As shown in Fig. 5d, tumor volumes and weights were reduced after treatment with different formulations, especially in the MnO₂/Co-DA@BiTE/HPT+L group. Moreover, the MnO₂/Co-DA@BiTE/HPT+L group inhibited tumor proliferation and induced more tumor necrosis and cell apoptosis (Supplementary Fig. 46). And the body weight of the mice during the treatment process showed no obvious change (Fig. 5e). Next, cytotoxic T lymphocytes from distant tumors were analyzed (Fig. 5f). The highest frequency of CD8⁺ T cells was observed in the MnO₂/Co-DA@BiTE/HPT+L group (33.99% in distant tumors). The immunofluorescence staining of CD4⁺ and CD8⁺ T cells in tumors displayed the same trend (Fig. 5g). Similarly, a significant increase (39.31% in distant tumors) in tumor-infiltrating mature DCs (CD80⁺CD86⁺) was detected in the MnO₂/Co-DA@BiTE/HPT+L group relative to that in the NS group (Fig. 5h and Supplementary Fig. 47). Additionally, after treatment with MnO₂/Co-DA@BiTE/HPT+L, the lowest frequency of Treg cells was observed in distant tumors (Fig. 5i and Supplementary Fig. 48). Next, the spleen and lymph nodes were collected to detect the maturation of DCs (Fig. 5j). Compared with the NS group, the MnO₂/Co-DA@BiTE/HPT+L group exhibited higher maturation of DCs. The spleens were collected to investigate the induction of Tem cells (Fig. 5k). Indeed, the number of Tems gated on CD3⁺CD8⁺ T cells only increased slightly after treatment with BiTE and MnO₂/Co-DA. Compared with the BiTE and MnO₂/Co-DA groups, the MnO₂/Co-DA@BiTE/HPT group displayed a much higher level of Tem, confirming that the combination of BiTE and MnO₂/Co-DA could induce immunological memory. Moreover, high secretion levels of proinflammatory cytokines in serum supported that MnO₂/Co-DA@BiTE/HPT+L induced a robust systemic immune response (Supplementary Fig. 49).

Antitumour efficacy in rechallenged and metastatic tumours

Inspired by the significant tumor therapy and immunostimulation efficacy of the synergistic treatment, the immune memory effect induced by MnO₂/Co-DA@BiTE/HPT was next investigated. We rechallenged the secondary CT26 tumors 11 days after resection of the primary CT26 tumors (Fig. 6a). The NS, BiTE, and MnO₂/Co-DA groups failed to avoid tumor recurrence due to weak immune memory effects. The progression of the secondary tumors was obviously suppressed in the MnO₂/Co-DA@BiTE/HPT+L group (Fig. 6b, c). The spleens and lymph nodes were collected to analyze Tem (Fig. 6d). After treatment with MnO₂/Co-DA@BiTE/HPT+L, the proportion of Tem cells in the spleen and lymph nodes was significantly increased, indicating the activation of the systemic immune response. In conclusion, the systemic antitumour immunity mediated by MnO₂/Co-DA@BiTE/HP+L could not only inhibit the growth of primary tumors, but also prevent tumor recurrence after surgery, which had significant therapeutic and preventive effects.

Finally, given the high immune memory of MnO₂/Co-DA@BiTE/HPT nanobombs, as demonstrated in the rechallenged model, we further evaluated the antimetastatic effect of MnO₂/Co-DA@BiTE/HPT in the lungs (Fig. 6e). Consistent with previous results of tumor volume, MnO₂/Co-DA@BiTE/HPT synergizing with PTT exerted a much greater antimetastatic effect (Fig. 6f). As shown in Fig. 6g, h, widespread metastatic nodules could be observed in the lung in the NS group, and the mice treated with BiTE and MnO₂/Co-DA exhibited lung metastatic nodules to some extent. Conversely, treatment with MnO₂/Co-DA@BiTE/HPT and MnO₂/Co-DA@BiTE/HPT+L efficiently inhibited the development of metastatic nodules in lung tissues. Moreover, H&E staining of lung sections further confirmed the antimetastatic effect of MnO₂/Co-DA@BiTE/HPT+L (Fig. 6i). MnO₂/Co-DA@BiTE/HPT+L not only increased the number of mature DCs in spleens and lymph nodes, but also significantly increased the amount of Tem. Such outstanding therapeutic benefits were likely attributed to a remarkable systemic immune response by the STING pathway, BiTE, and PTT therapy to prevent tumor metastasis.

Antitumor efficacy in the melanoma model

The therapeutic effects of MnO₂/Co-DA@BiTE/HPT were further evaluated in a melanoma model. Similarly, after treatment with MnO₂/Co-DA@BiTE/HPT+L, the mice displayed significant tumor growth inhibition (Fig. 7a–c). As shown in Fig. 7d, the mice treated with MnO₂/Co-DA@BiTE/HPT+L had the most marked tumor necrosis, cell apoptosis, and proliferation inhibition compared to the other groups. Subsequently, the immune cells (T cells and DCs) in tumor tissues were analyzed using flow cytometry. The percentages of both CD8⁺ T cells (Fig. 7e) and DCs maturation (Fig. 7f) were markedly greater in MnO₂/Co-DA@BiTE/HPT+L group than in the other treatment groups, suggesting that MnO₂/Co-DA@BiTE/HPT+L effectively activated the immune response, resulting in enhanced antitumor activity.

Biosafety evaluation

The biosafety of nanobombs is crucial in clinical applications. Thus, the body weight of the mice was monitored. As shown in Fig. 3e, the weight of tumor-bearing mice did not significantly change after treatment with different formulations. In addition, H&E staining was performed on the major organs (Supplementary Fig. 50). Compared with the NS group, no pathological changes were observed in the organs after treatment. The results showed that the nanobombs have great biological safety. The blood biochemical index is an important index to judge whether the body is healthy (Supplementary Fig. 51). The indices of liver and kidney function of tumor-bearing mice after administration were investigated. After treatment, there was no obvious difference in several indices compared with the NS group, indicating that the nanobombs would not destroy liver and kidney function at the experimental dose.

Hemolysis assay was used to evaluate the blood compatibility of MnO₂/Co-DA@BiTE/HPT nanobomb (Supplementary Fig. 52). The concentrations of MnO₂/Co-DA in MnO₂/Co-DA@BiTE/HPT nanobomb ranged from 25 to 200 μg/mL, while the hemolysis rates were below the safety standard of 5%, suggesting that MnO₂/Co-DA@BiTE/HPT exhibited excellent hemocompatibility.

To evaluate long-term safety, the mice were intravenously injected with normal saline and MnO₂/Co-DA@BiTE/HPT for 2 months (Supplementary Fig. 53a). The blood, kidneys, livers, lungs, hearts, and spleens of the mice were collected for assessment. Supplementary Fig. 53b shows the body weight drop for the treated mice in the early days after injection of MnO₂/Co-DA@BiTE/HPT, and the mice regained their body weights later. And the hematological tests and serum biochemical assays did not show toxicity of MnO₂/Co-DA@BiTE/HPT on blood cells and the function of the kidney, liver, and heart (Supplementary Fig. 53c–e). The HE-staining showed that MnO₂/Co-DA@BiTE/HPT did not harm the histological structure of the main organs (Supplementary Fig. 53f). Additionally, no significant change of cytokine levels, including IFN-γ, IL-6, IL-2, IL-10, and TNF-α, was observed (Supplementary Fig. 54). These results showed the long-term safety of MnO₂/Co-DA@BiTE/HPT.

Discussion

CRC is the third most common cancer after lung cancer and the second most deadly.⁴⁴ With the continuous progress of medical treatment, the methods for patients with CRC are more diverse. In addition to traditional radiotherapy and chemotherapy, targeted therapy and immunotherapy have ushered in increasingly better treatment options for CRC patients.⁴⁵ As an important part of immunotherapy, bispecific antibodies are only four types on the global market, and no bispecific antibodies against CRC have been approved. BiTE enhances the cytotoxicity of T cells by targeting CD3 and tumor-specific antigens.⁴⁶ However, the number of T cells in the TME is limited, and tumor cells can activate a variety of mechanisms to escape T-cell killing. The expression of some TAAs is universal, which may result in the “on-target off-tumor” effect of BiTE in practical applications and produce toxic reactions such as cytokine storms and tissue damage.¹⁰ Additionally, the immunosuppressive TME and the loss of target antigens may cause tumor immune tolerance to BiTE.⁸ Combination therapy has been extensively studied to reverse the microenvironment, break immune tolerance, and improve the efficacy of BiTE immunotherapy.^8,12

The cGAS-STING pathway can sense not only exogenous danger signals from viruses and bacteria, but also endogenous signals from damaged mitochondria and endocytosis fragments of dead cells.^14,16 Activation of the STING pathway in APCs may produce IFN-I to recruit and cross-activate effector T cells, thereby overcoming the “cold” tumor barrier.^19,20 To date, most of the Mn-based nanoparticles, such as MnO₂, MnSe₂, Mn₃O₄, Mn-MOF, and Mn-phenolic, have been reported and applied in STING-related immunotherapy.^47,48,49,50 However, the immunotherapy that relies on the activation of the STING pathway by Mn²⁺ cannot effectively inhibit tumor growth. In our study, the biometallic nanobombs were synthesized based on MnO₂ and metal-phenolic (Co-DA), which could activate the STING pathway by Mn²⁺/Co²⁺ and the damaged dsDNA of PTT. Aptamers, known as chemical antibodies, are structured oligonucleotide sequences that can mimic antibodies by folding into unique 3D structures to selectively bind to targets. To enhance the active targeting of STING agonist and BiTE, a PD-L1 aptamer was introduced in our work.

Herein, we prepared biometallic nanobombs (MnO₂/Co-DA@BiTE/HPT), which were based on the rational combination of STING activator, BiTE therapy, and PTT to significantly augment antitumour immunotherapy. The MnO₂/Co-DA nanobombs rapidly depolymerized under acidic conditions, releasing Co²⁺ and Mn²⁺. In addition to activating the STING pathway, MnO₂/Co-DA nanobombs had PTT functions under 808 nm laser irradiation. The triple-killing of MnO₂/Co-DA@BiTE/HPT nanobombs induced CT26 cell death to release damaged dsDNA, which could cooperate with bimetallic ions (Mn²⁺ and Co²⁺) to amplify the signals of the STING pathway, induce the maturation of BMDCs, and release IFN-β. Additionally, MnO₂/Co-DA@BiTE/HPT could induce initial T-cell proliferation and activation to enhance the antitumour effect. STING activator and BiTE were synchronously and accurately delivered to the tumor tissues with the targeting of HPT. There are limitations to our study. One of the major translational barriers is the scalability and reproducibility of our nanobombs. The current preparation process of MnO₂/Co-DA@BiTE/HPT is complex and time-consuming. Achieving consistent quality and yield on a large scale is a significant challenge. Variations in reaction conditions, such as temperature, pH, and reagent concentrations, can lead to differences in the properties of the final product, affecting its efficacy and safety. Another critical issue is the safety profile of our nanobombs in clinical applications. Although our preclinical studies have shown no obvious signs of severe toxicity, the long-term effects and potential side effects in humans are still unknown. The Mn²⁺/Co²⁺ and BiTE may trigger unexpected immune responses or interact with other drugs or medical conditions in patients. Extensive clinical trials and long-term follow-up studies are required to fully assess the safety and tolerability of our approach. Furthermore, the animal models in our study did not fully recapitulate the genetic, immunological, and microenvironmental heterogeneity observed in human colorectal malignancies. This limitation inherently restricts the generalizability of the efficacy data and might result in an overestimation of clinical translatability.

Bimetallic ions (Mn²⁺ and Co²⁺) activate the STING pathway with the assistance of PTT, transforming “cold” tumors into “hot” tumors, thereby overcoming the resistance of “cold” tumors to BiTE. Specifically, the activation of DCs induced by Mn²⁺/Co²⁺ and released dsDNA produced type I interferons and pro-inflammatory cytokines, which assessed and promoted T-cell proliferation, differentiation, and activation.²⁵ Activated DCs upregulated the expression of co-stimulatory molecules (such as CD80 and CD86), providing a second signal for T cell activation, thereby enhancing the cytotoxicity of BiTE-mediated T cells.^51,52 Furthermore, BiTE-mediated cytotoxicity of T cells led to the lysis and death of target cells, thereby releasing more tumor antigens that can be taken up and processed by DCs, further activating the STING signaling pathway, generating more type I interferons and pro-inflammatory cytokines, forming a positive feedback loop, and enhancing the anti-tumor immune response.

This study combined the triple-kill pathways of the STING pathway, BiTE therapy, and PTT for the first time in colorectal cancer immunotherapy, initiating both natural immunity and adaptive immunity, improving the immune microenvironment, producing long-term immune memory, inhibiting tumor growth, and preventing tumor recurrence and metastasis. This study is expected to provide a new idea for the treatment of colorectal cancer.

Materials and methods

Materials

Hyaluronic acid (HA, Mw~10 kDa) was obtained from Bloomage Freda Biopharm, Co. Ltd. The PD-L1 aptamer was obtained from Tsingke Biotechnology. KMnO₄, CoCl₂•6H₂O, NaCl, CaCl₂•2H₂O, MnCl₂•2H₂O, and CeCl₃•7H₂O were purchased from Aladdin. The calcein-AM/PI kit, JC-1 kit, DAPI, RIPA lysis buffer, and BCA kit were purchased from Beyotime Biotechnology. The γ-H2AX antibody was purchased from Abcam. HMGB1, CRT, and p-IRF3 antibodies were obtained from Huabio. p-TBK1 and p-STING antibodies obtained from CST. Picogreen dsDNA quantitation reagent and goldband 3-color regular range protein marker (10–180 kDa) were purchased from Yeasen. IL2, IL15, GM-CSF, IL4, CD3/CD28, and flow antibodies purchased from Biolegend (Table S1). Endo-free Plasmid Mini Kit was obtained from Omega. The IFN-β and IFN-γ ELISA kit was obtained from RuixinBio (Quanzhou, China). PD-L1/CD3 BiTE was obtained from Aiping Tong group. Serum was purchased from NEWZERUM (FBS-UP500). RPMI 1640 (L210KJ) and penicillin-streptomycin combination (S110JV) were obtained from BasalMedia.

Preparation and characterization of MnO₂/Co-DA@BiTE/HPT nanobombs

Then, 2 mL KMnO₄ (3.2 mg/mL) was added to a silene bottle to stir for 15 min. Then, 200 μL polyacrylamide hydrochloride (PAH) (37.4 mg/mL) was added and stirred at room temperature for 2 h to obtain MnO₂ nanoparticles.

The MnO₂ nanoparticles obtained above were added to the silene bottle after suspension, and 100 μL CeCl₃•6H₂O, CaCl₂•2H₂O, NaCl, and CoCl₂•6H₂O (20 mM) were added to the stirring for 15 min. Then, 20 μL DA (2 mg/mL) was added to the above solutions with stirring for 30 min to obtain MnO₂/Na-DA, MnO₂/Ca-DA, MnO₂/Ce-DA, and MnO₂/Co-DA.

MnO₂/Co-DA nanoparticles were suspended in saline, and the BiTE antibody was added and incubated at 4 °C for 24 h. Next, the material HPT was added to stir for 2 h in an ice bath. Finally, MnO₂/Co-DA@BiTE/HPT nanobombs were obtained by centrifugation.

In vitro release behavior of BiTE

To detect the drug loading of BiTE in MnO₂/Co-DA@BiTE/HPT nanobombs, an ultrafiltration tube (300 kDa) was used to remove the unloaded BiTE. Hyaluronidase (HAase) (500 μ/mL) was coincubated with MnO₂/Co-DA@BiTE/HPT to release the BiTE antibody. A PD-L1 ELISA kit was applied to detect the content of loaded BiTE.

MnO₂/Co-DA@BiTE/HPT nanobombs were transferred into a dialysis bag (MWCO: 300 kDa), which was immersed in PBS media containing 0 μ/mL, 200 μ/mL, and 500 μ/mL HAse. The released media were removed at different time points, and the same volume of fresh buffer was added. The cumulative release of BiTE was detected by a PD-L1 ELISA kit.

The function of the BiTE assay

T-cell/CT26 cells (4:1) were seeded in a 24-well plate overnight and cultured with IgG, BiTE, and MnO₂/Co-DA@BiTE/HPT (after culture with hyaluronidase) for 24 h. Next, the cells were blocked with 2% BSA buffer for 15 min and stained with anti-CD3, anti-CD8, anti-CD25, and anti-CD69 antibodies for 30 min for flow cytometry analysis (ACEA, Novocyte). The cell supernatant was analyzed with an IFN-γ ELISA kit.

Additionally, to investigate the effect of the 808 nm laser on BiTE biological activity, after laser irradiation, the binding affinity of PD-L1 in MnO₂/Co-DA@BiTE/HPT nanobombs was examined. First, the MnO₂/Co-DA@BiTE/HPT nanobombs were treated with 0.75 W cm⁻² and 1.5 W cm⁻² 808 nm lasers for 5 min. Next, the MnO₂/Co-DA@BiTE/HPT nanobombs were treated with hyaluronidase to release BiTE. Finally, the binding affinity of BiTE was evaluated via a PD-L1 ELISA kit.

In vitro ICD induction

CT26 cells were seeded in a 24-well plate coated with a cover glass and cultured overnight. The cells were incubated with PBS, BiTE, MnO₂/Co-DA, MnO₂/Co-DA+L, MnO₂/Co-DA@BiTE/HPT, and MnO₂/Co-DA@BiTE/HPT+L for 4 h and then irradiated with 808 nm laser (0.75 W cm⁻², 5 min). After culturing for another 8 h, CRT exposure and HMGB1 release were analyzed through immunofluorescence staining. The cells were fixed with 4% PFA for 15 min and blocked with 2% BSA for 1.5 h. Next, the cells were incubated with anti-CRT and anti-HMGB1 (1:200, Huabio) overnight. Finally, the cells were stained with Alexa Fluor 594-labelled goat-anti-mouse antibody (1:200, Huabio) for 2 h and DAPI for 5 min for observation with CLSM (Zeiss, LSM880).

Analysis of BMDC-stimulated maturation

CT26 cells were seeded in 24-well plates for 24 h and incubated with PBS, BiTE, MnO₂/Co-DA, MnO₂/Co-DA+L, MnO₂/Co-DA@BiTE/HPT, and MnO₂/Co-DA@BiTE/HPT+L. The cells were incubated with MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT nanobombs for 4 h under an 808 nm laser at 0.75 W cm⁻² for 5 min. Next, the cells were cultured for 24 h and stained with anti-CD11c, anti-CD80, and anti-CD86 antibodies for flow cytometry analysis. The cell supernatant was collected for the IFN-β assay. For the transwell experiment, CT26 cells were seeded on the transwell upper chamber for 24 h. The cells were incubated with PBS, BiTE, MnO₂/Co-DA, MnO₂/Co-DA+L, MnO₂/Co-DA@BiTE/HPT, and MnO₂/Co-DA@BiTE/HPT+L. The cells were incubated with MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT nanobombs for 4 h, under 808 nm laser at 0.75 W cm⁻² for 5 min. Then, BMDC cells were inoculated in the lower chamber for 24 h. Finally, the BMDCs were stained with anti-CD11c, anti-CD80, and anti-CD86 antibodies for flow cytometry analysis. The cell supernatant was collected for the IFN-β assay.

Stimulation of the STING pathway assay

CT26 cells were planted in culture overnight and treated with PBS, BiTE, MnO₂/Co-DA, MnO₂/Co-DA+L, MnO₂/Co-DA@BiTE/HPT, and MnO₂/Co-DA@BiTE/HPT+L. The cells were incubated with MnO₂/Co-DA and MnO₂/Co-DA@BiTE/HPT nanobombs for 4 h under an 808 nm laser at 0.75 W cm⁻² for 5 min. After that, the cell supernatant was collected for the following experiment. Next, DC2.4 and RAW264.7 cells were plated in culture dishes overnight. After adding the collected supernatant, the DC2.4 and RAW264.7 cells were cultured for 24 h, and the cells were collected for western blot assays.

In vitro antitumour immune system assay

T cells/BMDCs/CT26 (20:10:1) cells were seeded in 12-well plates overnight. And CT26 cells were labelled with CFSE. The supernatant of CT26 cells treated with different formulations was added to the coculture system for 24 h. Finally, the cells were collected for the CFSE⁺CT26 assay by flow cytometry.

In vivo antitumour effect assay

To investigate the in vivo antitumour effect of MnO₂/Co-DA@BiTE/HPT nanobombs, subcutaneous CT26 tumor-bearing BALB/c mice were established. When the tumor volume reached 200 mm³, the mice were randomly assigned to six groups and received different treatments on days 1 and 3, including Control (NS), BiTE (0.5 mg/kg), MnO₂/Co-DA (2 mg/kg), MnO₂/Co-DA+L, MnO₂/Co-DA@BiTE/HPT (MnO₂/Co-DA: 2 mg/kg, BiTE: 0.5 mg/kg), and MnO₂/Co-DA@BiTE/HPT+L. Two groups received 808 nm laser irradiation (0.75 W cm⁻², 5 min) at 12 h postinjection. The tumor volume and body weights were monitored every 2 days. On day 18, the mice were sacrificed. The tumors and the major organs were collected for H&E and immunofluorescence staining.

A CT26 bilateral-tumor model was established by inoculating 1 × 10⁶ and 5 × 10⁵ cells into the right and left flanks, respectively. When the tumor volume reached 200 mm³, the mice were treated as described above. The tumor volume was monitored.

To evaluate the effectiveness of MnO₂/Co-DA@BiTE/HPT nanobombs against lung metastasis, CT26 cells (1 × 10⁵) were intravenously injected after establishing primary tumors on day 7, and the treatments were performed according to the same method described above. When the mice died in the NS group, all mice were sacrificed, and the lungs were collected, and the number of nodules was recorded. In addition, H&E staining was performed.

To verify the prevention of postoperative recurrence, a CT26 tumor-bearing mouse model was first established. When the tumor volume reached 100 mm³, the mice were randomly divided into six groups and treated with different formulations. On day 10, the tumor was surgically removed. On day 21, CT26 cells (1 × 10⁶) were inoculated in the left flank. The tumor volume of the mice was recorded.

Evaluation of antitumour immune response

To analyze T-cell infiltration, CT26 tumor tissues were collected and then digested with collagenase (1 mg/mL)/hyaluronidase (100 μg/mL)/DNAse I (100 μg/mL) to obtain single-cell suspensions. The single-cell suspensions were blocked with 2% BSA and stained with Zombie Red™ Fixable Viability Kit, anti-CD45, anti-CD3, anti-CD4, and anti-CD8 antibodies for 30 min. Next, the cells were fixed with 4% PFA for 15 min to be analyzed with flow cytometry. To analyze the Treg cells, after blocking, the single-cell suspensions were stained with the Zombie Red™ Fixable Viability Kit, anti-CD45, anti-CD3, anti-CD4, and anti-CD25 antibodies for 30 min. Next, the cells were immobilized and permeated. Finally, the cells were stained with anti-FOXP3 antibodies for 30 min for analysis by flow cytometry. Additionally, the single-cell suspensions were stained with Zombie Red™ Fixable Viability Kit, anti-CD45, anti-CD11c, anti-CD80, and anti-CD86 antibodies for 30 min. Finally, the cells were fixed with 4% PFA to analyze the mature DCs by flow cytometry.

To analyze effector T cells (Tem) in the spleen, spleen and lymph node single-cell suspensions were obtained and stained with anti-CD3, anti-CD8, anti-CD44, and anti-CD62L antibodies for flow cytometry assays (CD3⁺CD8⁺CD44⁺CD62L⁻).

To analyze mature DCs in lymph nodes, the lymph nodes were collected to obtain single-cell suspensions. Then, the single-cell suspensions were stained with anti-CD11c, anti-CD80, and anti-CD86 antibodies for flow cytometry assays (CD11c⁺CD80⁺CD86⁺).

The antitumor efficacy in melanoma model

The therapeutic effects of MnO₂/Co-DA@BiTE/HPT were further evaluated by a subcutaneous B16-F10 (5 × 10⁵ cells) tumor-bearing model. When the tumor volume reached 100 mm³, the mice were randomly assigned to six groups and received different treatments on days 1 and 3, including Control (NS), BiTE (0.5 mg/kg), MnO₂/Co-DA (2 mg/kg), MnO₂/Co-DA+L, MnO₂/Co-DA@BiTE/HPT (MnO₂/Co-DA: 2 mg/kg, BiTE: 0.5 mg/kg), and MnO₂/Co-DA@BiTE/HPT+L. Two groups received 808 nm laser irradiation (0.75 W cm⁻², 5 min) at 12 h postinjection. The tumor volume and body weights were monitored every 2 days. On day 8, the mice were sacrificed. The tumors were collected for H&E, TUNEL, and Ki67 staining. Additionally, a portion of the tumors was used for flow cytometry analysis.

Statistical analysis

The experimental data are shown as the mean ± standard deviation (SD). The differences between multiple groups were performed using one-way analysis of variance (ANOVA) with Tukey’s test. The student’s test was used to analyze the significant differences between the two groups. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Data availability

All data generated or analyzed during this study have been included either in this article or in the supplementary information files. The data reported in this paper have been deposited in the OMlX, China National Center for Bioinformation/Belling instituteof Genomics, Chinese Academy of Sciences (https:/ingdc.cncb.ac.cn/omix: accession no. OMIX013900).

References

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Acknowledgements

This work was financially supported by the National Natural Sciences Foundation of China (31971308, 81960769, and U1903211), National S&T Major Project (2019ZX09301-147), Noncommunicable Chronic Diseases-National Science and Technology Major Project (2023ZD050610X/2023ZD0506100), China Postdoctoral Science Foundation (2024M762225), and Sichuan Science and Technology Program (2022YFS0007 & 22GJHZ0015).

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Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China
Min Mu, Hui Li, Bo Chen, Rangrang Fan, Kunhong Zhong, Chenqian Feng, Gang Guo & Aiping Tong
Department of Laboratory of Cancer Therapy and Immunity, Department of Radiation Oncology and Department of Head and Neck Oncology, West China Hospital, Sichuan University, Chengdu, China
Min Mu, Nianyong Chen & Bingwen Zou
Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu, China
Guoqing Wang

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Min Mu
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Hui Li
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Chenqian Feng
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Gang Guo
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Contributions

M.M. designed and performed the experiments, analyzed the data, and prepared the manuscript. H.L. collected the data. B.C. performed characterization of the nanobombs. R.F. and G.W. provided suggestions and technical support. K.Z. provided the plasmid. H.L., B.C., and C.F. assisted in the in vivo experiments. G.G. conceptualized, supervised, and supported the whole project. A.T. edited manuscript and supervised the study. N.C. supervised and supported the study. B.Z. conceptualized, supervised, and supported the whole project. All the authors have read and approved the article.

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Correspondence to Gang Guo, Aiping Tong or Bingwen Zou.

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Mu, M., Li, H., Chen, B. et al. Unleashing the potential of bimetallic nanobomb-mediated STING pathway to enhance bispecific T-cell engager against colorectal cancer photo-immunotherapy. Sig Transduct Target Ther 11, 80 (2026). https://doi.org/10.1038/s41392-026-02596-6

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Received: 18 May 2025
Revised: 22 November 2025
Accepted: 14 January 2026
Published: 05 March 2026
Version of record: 05 March 2026
DOI: https://doi.org/10.1038/s41392-026-02596-6