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. 2026 Aug 11;40:103550. doi: 10.1016/j.mtbio.2026.103550

Biohybrid Salmonella typhimurium for synergistic chemoimmunotherapy via localized chemotherapy and in situ PD-L1 blockade

Longxue Guan a,b,1, Qinxiao Sun b,1, Hancheng Jiang c,1, Dandan Liang b, Shiqi Zhang b, Laili Huang b, Xingang Guan a,⁎, Xiuli Hu c,⁎⁎, Guofu Chen a,⁎⁎⁎
PMCID: PMC13487396  PMID: 42621362

Abstract

Chemotherapy can substantially enhance the efficacy of cancer immunotherapy; however, the clinical translation of chemoimmunotherapy is frequently hampered by inefficient drug delivery and dose-limiting systemic toxicity. To address these challenges, we engineered a tumor-targeting Salmonella typhimurium–based platform (VNP@PD-L1nb/DOX) that simultaneously enables localized chemotherapy and in situ PD-L1 blockade. The attenuated Salmonella typhimurium strain VNP20009 was genetically engineered to secrete PD-L1-targeting nanobodies (PD-L1nb), and surface-functionalized with a phenylboronic acid (PBA)-conjugated doxorubicin prodrug (DOX-PBA) via reversible boronate ester bonds. This design allows hydrogen peroxide (H2O2)-responsive release of active doxorubicin within the tumor microenvironment while maintaining negligible toxicity toward normal tissues and the bacterial carrier itself. The engineered bacteria exhibited pronounced tumor-selective accumulation and continuously secrete PD-L1nb into the extracellular space for PD-L1 selective blockade. In a murine triple-negative breast cancer (TNBC) model, VNP@PD-L1nb/DOX significantly suppressed tumor growth, induced immunogenic cell death (ICD), and promoted robust systemic immune activation. The ICD-driven immune priming, together with localized PD-L1 blockade, synergistically enhanced intratumoral infiltration and activation of CD8+ T cells while concurrently depleting regulatory T (Treg) cells. Collectively, our findings underscore the promise of engineered bacterial vectors as a versatile integrated platform for targeted chemoimmunotherapy in advanced solid tumors.

Keywords: Salmonella typhimurium, Chemoimmunotherapy, PD-L1 nanobody, Drug delivery, Antitumor immunity

Graphical abstract

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Highlights

  • •

    VNP@PD-L1nb shows tumor-selective accumulation and sustained PD-L1nb secretion.

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    VNP@PD-L1nb/DOX enables localized chemotherapy and in situ PD-L1 blockade.

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    VNP@PD-L1nb/DOX achieves potent tumor suppression with minimal systemic toxicity.

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    Biohybrid system triggers potent antitumor immunity for enhanced chemoimmunotherapy.

1. Introduction

Cancer immunotherapy has revolutionized cancer treatment by harnessing the body's immune system to recognize and eliminate malignant cells [[1], [2], [3]]. Immune checkpoint inhibitors (ICIs), particularly antibodies targeting the PD-1/PD-L1 axis, have shown remarkable clinical success in treating many advanced cancers [4,5]. However, only a minority of patients respond to ICI monotherapy due to the poor tumor immunogenicity and immunosuppressive tumor microenvironment (TME) [[6], [7], [8]]. Chemotherapy can not only kill tumor cells and release tumor-associated antigens, but also elicit antitumor immunity by inducing ICD [9,10]. Despite the significant promise, the clinical translation of chemoimmunotherapy faces significant hurdles, primarily stemming from poor drug delivery efficiency and systemic toxicities associated with chemotherapeutics and ICIs [11,12]. This rationale design of novel formulations for maximizing therapeutic synergy while minimizing off-target effects holds promise to overcome the current limitations of chemoimmunotherapy [13,14].

Bacteria, naturally occurring microorganisms with single cells, have been used as therapeutic agents to treat cancers for more than a century [[15], [16], [17]]. They were first employed for cancer treatment in the late 19th century. William B. Coley demonstrated that intratumoral inoculation with Streptococcus pyogenes and Serratia marcescens could result in tumor regression, laying the foundation for bacterial-based cancer therapy [18,19]. Certain facultative and obligate anaerobic bacterial strains specifically migrate to tumors and preferentially proliferate in tumors, leading to the tumor demission [[20], [21], [22]]. Despite the limited cytotoxic potency of natural bacteria, synthetic biology could improve the bacteria's therapeutic efficacy and minimize their side effects [23,24]. Bacteria can be engineered to express and release genetically encoded therapeutic cargoes in response to external stimuli (et al., elevated temperature [25], ultrasound [26], magnetic field [27], and quorum signals [28]) and intrinsic tumor microenvironmental cues (including lactate accumulation, acidic pH, and hypoxia) [[29], [30], [31]]. The engineered microbes functioned as “smart factories” for precision cancer therapy. In addition to producing endogenous agents, engineered microbes can serve as active delivery systems to efficiently deliver exogenous payloads to potentiate chemotherapy [[32], [33], [34]], photothermal therapy [35,36], photodynamic therapy [37,38], immunotherapy [39,40], and combination treatment [[41], [42], [43]]. However, the precise spatiotemporal control of therapeutic cargo release without off-target activation remains technically challenging.

Despite the development of numerous engineered bacteria for immunotherapy, bacterial-based platforms for chemoimmunotherapy remain scarce. The substantial scientific and engineering hurdles include the challenge of engineering a single bacterial system to both deliver chemotherapeutic drugs and produce immunomodulatory agents in a coordinated and controllable manner. Furthermore, achieving effective chemotherapeutic drug loading and precise release in tumors without compromising the bacteria's own viability and tumor-targeting ability remains a significant obstacle. To address this gap, we present a Salmonella typhimurium–based platform (VNP20009) co-functionalized with surface-conjugated doxorubicin prodrug (DOX-PBA) and genetically engineered to express and secrete PD-L1nb, yielding the multifunctional construct VNP@PD-L1nb/DOX (Scheme 1). The engineered bacteria stably secreted PD-L1nb into the extracellular milieu, enabling selective binding to PD-L1 on tumor cells. A phenylboronic acid (PBA)-functionalized doxorubicin prodrug (DOX-PBA) was selected to form reversible boronate ester bonds with polysaccharides on the bacterial surface [[44], [45], [46]], thus facilitating a hydrogen peroxide (H2O2)-responsive and liberation of active DOX within the tumor microenvironment. Furthermore, the PBA modification renders the prodrug non-toxic to healthy tissues and the bacterial carrier itself. The integration of localized chemotherapy with in situ PD-L1 checkpoint blockade effectively reprogrammed immunologically “cold” tumors into “hot” tumors. In a triple-negative breast cancer (TNBC) mouse model, VNP@PD-L1nb/DOX significantly suppressed tumor growth and elicited a robust antitumor immune response, showing the promising potential as a potent chemoimmunotherapeutic strategy for TNBC.

Scheme 1.

Scheme 1

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Schematic illustration of the fabrication of VNP@PD-L1nb/DOX and its synergistic chemoimmunotherapy mechanism. (A) Construction of the dual-functional engineered bacteria. The attenuated VNP20009 strain is genetically engineered to secrete PD-L1nb and surface-functionalized with DOX-PBA prodrugs to form VNP@PD-L1nb/DOX. (B) Synergistic antitumor mechanism. Upon tumor colonization, the engineered bacteria responsively release DOX to induce ICD. Concurrently, the bacterially secreted PD-L1nb blocks the PD-L1 immune checkpoint, leading to a synergistic enhancement of antitumor immunity.

2. Results and discussion

2.1. Preparation and characterization of VNP@PD-L1nb

To enable extracellular export of expressed protein, a Salmonella secretion signal peptide (SP) was fused to the N-terminus of PD-L1nb in this study (Fig. 1A). The sequenced recombinant plasmid was transformed into Salmonella VNP20009 competent cells to generate the engineered strain, designated VNP@PD-L1nb. VNP@PD-L1nb exhibited a size (Support Information, Fig. S1), surface charge (Fig. 1B) and morphology (Fig. 1C and Support Information, Fig. S2) comparable to the parental strain. Western blotting confirmed successful expression and secretion of PD-L1nb, as evidenced by anti-His signals in both bacterial lysates and culture supernatants (Fig. 1D, and Support Information Fig. S3). Following L-arabinose induction, the strain exhibited sustained protein production, with robust secretion detectable as early as 10 h post-induction (Support Information, Fig. S4). ELISA results showed that the concentration of PD-L1nb in the culture supernatant reached 84.64 ng/mL after 16 h of induction.

Fig. 1.

Fig. 1

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Preparation and characterization of VNP@PD-L1nb. (A) Schematic of VNP@PD-L1nb preparation using VNP20009. (B) Surface charge analysis of engineered bacteria. (C) Morphology analysis of VNP@PD-L1nb by TEM imaging. Scale bar: 500 nm. (D) Western blot analysis of supernatant (SN) and cell pellet of VNP@PD-L1nb using anti-His antibody. (E) Cell compatibility analysis of VNP@PD-L1nb in HEK293T cells via CCK-8 assay. (F and G) PD-L1 blockade analysis of purified PD-L1nb and concentrated SN. The data are presented as mean ± SD. n = 3. ns, not significant. ***p < 0.001. ****p < 0.0001. Statistical significance was calculated by one-way analysis of variance (ANOVA) followed with Tukey's post hoc test. Pretreatment of PD-L1nb or SN significantly decreased the binding of PE-αPD-L1 with breast cancer cells. (H) In vivo distribution of VNP@PD-L1nb and BL21 cells transformed with luciferase plasmid using an IVIS for bioluminescence in vivo seven days post injection. (I) Immunostaining analysis of PD-L1nb in tumors using anti-His antibodies. Nucleus (Blue), PD-L1-His protein (Red). Scale bar is 100 μm.

The biocompatibility of the engineered bacteria was assessed via CCK-8 assay in HEK293T cells. Neither VNP20009 nor VNP@PD-L1nb exhibited obvious cytotoxicity at the tested concentrations, demonstrating good cellular compatibility (Fig. 1E). Furthermore, PD-L1nb was purified from the culture supernatant using Ni-NTA affinity chromatography (Support Information Fig. S5). The PD-L1 binding ability of both the purified PD-L1nb and the concentrated bacterial supernatant (SN) was evaluated by competitive binding assays using PE-conjugated anti–PD-L1 antibody (PE-αPD-L1). Pretreatment of 4T1 cells with either purified PD-L1nb or bacterial SN significantly reduced PE-αPD-L1 binding (Fig. 1F and G), confirming that the secreted PD-L1nb in SN effectively blocks the PD-L1 receptor on tumor cells.

To validate the tumor-targeting capability of engineered bacteria, we established a subcutaneous TNBC mouse model and evaluated the biodistribution of VNP@PD-L1nb, using Escherichia coli BL21 as a control. Both bacteria were transformed with a luciferase-expressing plasmid to generate bioluminescent reporter strains for tumor imaging. Mice were intravenously administered 1 × 107 CFU of BL21 cells or VNP@PD-L1nb, and in vivo bioluminescence imaging was performed seven days post-inoculation. Strong luminescence signals were detected specifically in the tumors of mice treated with engineered VNP, whereas a negligible signal was observed in the tumors of the BL21 group (Fig. 1H). Notably, in contrast to the pronounced accumulation of BL21 in the liver and kidneys, VNP@PD-L1nb displayed highly selective tumor localization with minimal off-target distribution (Support Information, Fig. S6). To directly detect local therapeutic exposure in vivo, we further examined PD-L1nb levels in tumor tissues by immunostaining. Three days after VNP@PD-L1nb injection, tumors were excised and stained with anti-His antibodies to detect His-tagged PD-L1nb. The results showed strong intratumoral PD-L1nb signals (Fig. 1I), indicating that the engineered bacteria were able to produce PD-L1nb locally within the tumor microenvironment. This localized PD-L1nb production may provide sustained checkpoint blockade while reducing the need for high systemic antibody exposure. These findings highlight the robust tumor-homing capacity and sustained local PD-L1nb production of our engineered bacteria, supporting their potential as a tumor-targeted delivery platform for checkpoint blockade.

2.2. Preparation and characterization of VNP@PD-L1nb/DOX

Building upon VNP@PD-L1nb, we next designed a chemotherapeutic-loaded bacterial platform (VNP@PD-L1nb/DOX) by conjugating a phenylboronic acid–modified doxorubicin prodrug (DOX-PBA) to the bacterial surface (Fig. 2A). Leveraging the abundant sialoglycoproteins and polysaccharides present on the bacterial envelope, DOX-PBA can be covalently anchored via pH-sensitive phenylboronate ester (PBE) linkages (Fig. 2B). This surface drug conjugation preserved the rod-like morphology of the bacteria (Fig. 2C) and resulted in a modest increase in hydrodynamic diameter (Fig. 2D). Flow cytometry analysis confirmed the successful decoration of DOX-PBA on engineered bacteria (Fig. 2E). The zeta potential of VNP@PD-L1nb/DOX was measured to be −21.4 mV. Confocal laser scanning microscopy (CLSM) imaging demonstrated the successful decoration of DOX-PBA on the surface of engineered bacteria (Fig. 2F). Z-stack scans showed that the red fluorescence was mainly observed in the focal planes containing rod-shaped bacteria and appeared preferentially enriched at the bacterial periphery (Support Information, Fig. S7A). Three-dimensional reconstruction further demonstrated that the red fluorescence signal was predominantly distributed along the outer contour of the bacterial particles, supporting a mainly surface-associated localization (Support Information, Fig. S7B).

Fig. 2.

Fig. 2

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Preparation and characterization of VNP@PD-L1nb/DOX. (A) UV−vis absorption spectra of DOX and DOX-PBA. (B) Schematic illustration of the conjugation of DOX-PBA to surface-exposed glucose residues on bacterial cells. (C) Morphology analysis of VNP@PD-L1nb/DOX by TEM analysis. Scale bar: 200 nm. (D) Hydrodynamic size distribution determined by DLS. The data are presented as mean ± SD. n = 3. (E) Flow cytometry analysis showed successful decoration of DOX-PBA on bacterial cells. 4T1 cells were treated with 10 μM DOX-PBA for 2 h at 37 °C. (F) Fluorescence microscopy image of VNP@PD-L1nb/DOX using the inherent fluorescence of DOX. Scale bar: 2 μm. (G) Encapsulation efficiency analysis in engineered bacteria. (H) Hemolysis assay evaluating blood compatibility. (I) Stability analysis by measuring the surface charge over time. The data are presented as mean ± SD. n = 3. (J) Growth kinetics of VNP@PD-L1nb/DOX with varying concentrations of DOX-PBA. (K) Growth kinetics of VNP@PD-L1nb/DOX over time.

To confirm the chemical reaction between DOX-PBA and sugars, we mixed DOX-PBA with equal molar of glucose and added the mixed solution to cancer cells. The result demonstrated that reaction of DOX-PBA with VNP@PD-L1nb was substantially inhibited by the addition of glucose, showing the specific reaction of DOX-PBA with the glucose on the bacterial surface (Support Information, Fig. S7). Notably, the encapsulation efficiency (EE) of prodrug in bacteria exceeded 95% at all tested concentration, likely owing to the abundance of glucose and sialic acid on the bacteria's surface (Fig. 2G). Hemolysis assays indicated excellent blood compatibility of VNP@PD-L1nb/DOX (Fig. 2H). Furthermore, the bacaterial delivery system exhibited remarkable colloidal stability, with no significant change in surface charge observed over 15 days under physiological conditions (Fig. 2I). Bacterial proliferation was monitored by measuring optical density at 600 nm (OD600) over time. DOX-PBA decoration did not impair bacterial viability or proliferation at the employed concentration (Fig. 2J). Moreover, VNP@PD-L1nb, VNP@DOX-PBA, and VNP@PD-L1nb/DOX all exhibited growth kinetics comparable to those of the parental VNP20009 strain (Fig. 2K), indicating that genetic modification and surface conjugation did not compromise their intrinsic proliferative capacity.

We also examined whether DOX-PBA modification on the surface of VNP@PD-L1nb affected the tumor-targeting ability. The results showed that DOX-PBA decoration did not impair the tumor-homing capacity of VNP@PD-L1nb, supporting its potential as a targeted delivery platform for both checkpoint inhibitors and chemotherapeutic agents to enhance cancer treatment (Support Information, Fig. S8).

2.3. In vitro drug release and cytotoxicity analysis of VNP@PD-L1nb/DOX

Given the responsive release property of DOX prodrug (Fig. 3A), the drug release profile of VNP@PD-L1nb/DOX in vitro was evaluated using a dialysis method under mimic tumor microenvironment conditions. Notably, 85.7% of the total DOX was released in acidic PBS (pH 5.5) after 72 h of incubation, compared to only 25.4% in neutral PBS (pH 7.4), highlighting the acid-responsive release profile of VNP@PD-L1nb/DOX (Fig. 3B). Furthermore, the presence of H2O2 markedly accelerated drug release, confirming its H2O2-responsive drug delivery behavior (Fig. 3C). VNP@PD-L1nb/DOX also exhibited acid-sensitive drug release behavior in PBS containing 10% FBS (Support Information, Fig. S10). Cellular uptake of DOX-loaded bacteria was assessed in 4T1 breast cancer cells using both flow cytometry and CLSM imaging. Compared to free DOX-PBA, the VNP@PD-L1nb/DOX group exhibited a 2.1-fold increase in intracellular DOX fluorescence intensity (Fig. 3D and E), indicating enhanced cellular internalization mediated by the bacterial carrier. CLSM imaging further revealed the efficient internalization of VNP@PD-L1nb/DOX in 4T1 cells within 2 h of incubation (Fig. 3F). To evaluate the therapeutic efficacy of VNP@PD-L1nb/DOX, the cytotoxic analysis was employed via CCK-8 assay. Because viable VNP bacteria can proliferate in the tumor-cell compartment and interfere with cell-viability measurements, a Transwell system was used to physically separate bacteria from 4T1 cells while permitting diffusion of released DOX and secreted PD-L1nb. 4T1 cells were seeded in the lower chamber, while DOX-loaded bacteria were confined to the upper insert. Both VNP@DOX and VNP@PD-L1nb/DOX induced dose-dependent cytotoxicity against TNBC cells (Fig. 3G). Flow cytometry confirmed that DOX-decorated bacteria effectively induce apoptosis in tumor cells (Fig. 3H). These results underscore the potential of engineered therapeutic bacteria as effective platforms for cancer chemotherapy.

Fig. 3.

Fig. 3

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In vitro bioactivity analysis of VNP@PD-L1nb/DOX. (A) Proposed mechanism of the degradation of the DOX-PBA prodrug triggered by H2O2. (B) pH-dependent release profiles of DOX-PBA from bacteria in PBS at varying pH values. (C) In vitro H2O2-responsive DOX-PBA release profile. In vitro drug release was evaluated in PBS containing H2O2 (100 μM) or in H2O2-free PBS. (D) Quantitative analysis of intracellular DOX accumulation (n = 3). *p < 0.05. Statistical significance was calculated by one-way analysis of variance (ANOVA) followed with Tukey's post hoc test. (E) Cellular uptake of DOX-PBA, VNP@DOX-PBA, VNP@PD-L1nb/DOX assessed by flow cytometry. (F) CLSM images showing intracellular distribution of bacteria-mediated DOX delivery. Scale bar: 10 μm. (G) Cytotoxicity analysis of VNP@DOX-PBA and VNP@PD-L1nb/DOX against breast cancer cells evaluated by CCK-8 assay (n = 4). (H) VNP@PD-L1nb/DOX induced the apoptosis of cancer cells determined by flow cytometry.

2.4. In vivo antitumor efficacy of VNP@PD-L1nb/DOX in a TNBC mouse model

We next evaluated the in vivo antitumor efficacy of VNP@PD-L1nb/DOX in a 4T1 subcutaneous murine tumor model. Tumor-bearing mice were intravenously administered PBS, VNP, free DOX, VNP@PD-L1nb, VNP@DOX-PBA, or VNP@PD-L1nb/DOX via tail vein injection on days 6, 11, and 16 (Fig. 4A). Compared with the PBS group, free DOX, VNP@PD-L1nb, and VNP@DOX-PBA achieved tumor growth inhibition rates of 39.1%, 47.4%, and 56.2%, respectively, on day 18 (Fig. 4B). Notably, VNP@PD-L1nb/DOX exhibited the strongest antitumor efficacy, with a tumor growth inhibition rate of 73.3% (Fig. 4B). All treatment groups exhibited no significant body weight loss throughout the study, indicating favorable systemic tolerability (Fig. 4C). Ex vivo analysis of excised tumors further corroborated the superior therapeutic performance of VNP@PD-L1nb/DOX, as evidenced by markedly reduced tumor weight (Fig. 4D and E). H&E staining of tumor sections revealed extensive necrosis and structural disintegration in the VNP@PD-L1nb/DOX group, confirming potent tumor-killing activity (Fig. 4F). Moreover, Ki67 immunohistochemical staining showed a substantial decrease in proliferating tumor cells following chemoimmunotherapy (Fig. 4G). TUNEL analysis revealed substantial cell apoptosis in the tumors receiving the treatment of VNP@PD-L1nb/DOX (Fig. 4H), underscoring the ability of VNP@PD-L1nb/DOX to suppress cancer cell proliferation.

Fig. 4.

Fig. 4

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In vivo antitumor analysis of VNP@PD-L1nb/DOX in a subcutaneous TNBC mouse model. The mice were intravenously administered via tail vein injection on days 6, 11, and 16 post-tumor inoculation. (A) Schematic of tumor inoculation and treatment schedule. (B) Tumor growth curves over time (n = 5). (C) Bodyweight analysis (n = 5). (D) Quantification of tumor weight in different groups (n = 5). (E) Excised tumor images on day 18. G1: PBS; G2: VNP; G3: VNP@PD-L1nb; G4: VNP@DOX-PBA; G5: VNP@PD-L1nb/DOX. (F) H&E staining of tumor sections. Scale bar: 100 μm. (G) Ki-67 immunostaining of tumor tissues receiving different treatments. Scale bar: 100 μm. (H) TUNEL analysis in tumor tissues. Scale bar: 100 μm. Data in Fig. 4B, C, and 4E are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

2.5. VNP@PD-L1nb/DOX promoted ICD in tumors of mice bearing TNBC model

To confirm the successful secretion of PD-L1nb into the tumor microenvironment in vivo, we analyzed the expression of PD-L1nb in tumor lysates by western blot using anti-His antibodies. A distinct band corresponding to PD-L1nb was observed in tumors colonized by VNP@PD-L1nb and VNP@PD-L1nb/DOX (Fig. 5A, Support Information, Fig. S11). These results demonstrate that the engineered bacteria effectively colonize tumors and sustainably express and secrete PD-L1nb into the local microenvironment. To elucidate the mechanistic basis underlying the antitumor efficacy of VNP@PD-L1nb/DOX, we investigated its capacity to induce ICD. Western blot analysis using an anti–high-mobility group box B1 (HMGB1) antibody revealed that both VNP@DOX and VNP@PD-L1nb/DOX significantly promoted HMGB1 release into the tumor microenvironment (Fig. 5B and C, Support Information, Fig. S12). Additionally, immunofluorescence staining for CRT demonstrated pronounced CRT exposure on tumor cells following VNP@PD-L1nb/DOX treatment (Fig. 5D). Consistent with this, intracellular ATP levels were markedly reduced in tumors treated with DOX-loaded bacterial formulations, indicating active release of ATP into the extracellular space (Fig. 5E). Collectively, these findings indicate that VNP@PD-L1nb/DOX effectively triggers the release of canonical damage-associated molecular patterns (DAMPs), thereby converting immunologically “cold tumors” into “hot tumors” and priming antitumor immune responses. In addition, we evaluated the capacity of VNP@PD-L1nb/DOX to elicit systemic immune activation. Serum concentrations of IFN-γ and TNF-α were quantified by ELISA. Relative to the control group, our bacterial delivery platform induced a 2.35-fold enhancement in IFN-γ levels (Fig. 5F) and a 2.43-fold increase in TNF-α (Fig. 5G), demonstrating an induced immune response triggered by the bacterial therapeutic system.

Fig. 5.

Fig. 5

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VNP@PD-L1nb/DOX induces ICD in tumor cells and promotes systemic immune activation in vivo. G1: PBS; G2: VNP; G3: VNP@PD-L1nb; G4: VNP@DOX-PBA; G5: VNP@PD-L1nb/DOX. (A) Western blot analysis of PD-L1nb expression in tumor lysates using anti-His antibodies. (B) Western blot analysis of HMGB1 expression in tumor lysates using anti-HMGB1 antibodies. (C) Quantification analysis of HMGB1 expression (n = 3). (D) Immunohistochemical staining of calreticulin (CRT) exposure on tumor cells. (E) Quantification of intratumoral ATP levels (n = 5). (F) Serum concentration of interferon-gamma (IFN-γ) (n = 5). (F) Serum levels of tumor necrosis factor-alpha (TNF-α) in treated mice (n = 5). Data are presented as mean ± SD. Statistical significance was determined by using one-way analysis of variance with Tukey's test. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

2.6. VNP@PD-L1nb/DOX elicited a robust antitumor immune response in vivo

Inspired by the observed immune activation, we next examined the infiltration and activation of immune cells induced by VNP@PD-L1nb/DOX. Flow cytometric analysis of tumor-infiltrating lymphocytes revealed a significant increase in CD8+ T cell infiltration following treatment with VNP@PD-L1nb/DOX (Fig. 6A, Supporting Information, Figs. S13 and S17A). Critically, the frequencies of granzyme B+CD8+ T cells (Fig. 6B, Supporting Information, Figs. S14 and S17B) and perforin+CD8+ T cells (Fig. 6C, Supporting Information, Figs. S15 and S17C) were markedly elevated, indicating robust activation of cytotoxic effector functions. Given the inhibitory role of immunosuppressive Tregs on CD8+ T cells, we further assessed the percentage of Tregs in the tumor microenvironment. Compared with the modest Treg reduction in VNP@PD-L1nb and VNP@DOX-PBA group, VNP@PD-L1nb/DOX treatment reduced the Treg population by 62.25% compared to the PBS group, demonstrating the advantage of cooperative immune remodeling in alleviating immunosuppression and fostering antitumor immunity (Fig. 6D, Supporting Information, Figs. S16 and S17D). Immunofluorescence imaging of tumor sections revealed pronounced infiltration of CD8+ T cells in tumors treated with VNP@PD-L1nb/DOX (Fig. 6E). Together with complementary immunological analyses, these findings demonstrate that our engineered bacterial delivery system elicits a robust antitumor immune response.

Fig. 6.

Fig. 6

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VNP@PD-L1nb/DOX elicits a potent antitumor immune response in a TNBC mouse model. G1: PBS; G2: VNP; G3: VNP@PD-L1nb; G4: VNP@DOX-PBA; G5: VNP@PD-L1nb/DOX. (A) Flow cytometry analysis of tumor-infiltrating CD8+ T cells (n = 3). (B) Flow cytometry analysis of CD8+GzmB+ T cells in tumors (n = 3). (C) Flow cytometry analysis of CD8+PFP+ T cells in tumors (n = 3). (D) Flow cytometry analysis of Treg cells in the tumor microenvironment (n = 3). (E) Immunohistochemical staining of CD8+ T cell infiltration in tumor sections from different treatment groups.

We further evaluated the biodistribution and biosafety of VNP@PD-L1nb/DOX. Bacterial localization in major organs and tumors was assessed by plating tissue homogenates on selective agar plates. At 1 day post-injection, viable bacteria were detected in both healthy organs and tumors (Fig. 7A). In contrast, by day 14, no bacterial colonies were observed in the heart, spleen, lung, or kidney, whereas persistent colonization was evident exclusively in the tumor, confirming the platform's preferential tumor colonization. H&E staining of major organs (Fig. 7B), along with serum biochemical analysis of liver and kidney function (Supporting Information, Fig. S18), revealed no significant histopathological abnormalities or organ dysfunction throughout the treatment period, underscoring the favorable biosafety profile of VNP@PD-L1nb/DOX. Because attenuated Salmonella is intrinsically immunogenic, repeated intravenous administration may induce host anti-bacterial immune responses, which could accelerate bacterial clearance and affect the persistence of subsequent doses. In the present study, bacterial clearance kinetics and anti-Salmonella antibody titers after repeated administration were not systematically evaluated. Future studies will investigate these immunogenicity-related parameters to optimize dosing frequency, bacterial persistence, and therapeutic efficacy.

Fig. 7.

Fig. 7

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In vivo biosafety assessment of VNP@PD-L1nb/DOX. (A) Bacterial colonization in major organs and tumors at indicated time points post-injection, as determined by plating tissue homogenates on selective agar plates. (B) H&E staining of main organs and tumor sections from mice in different treatment groups. G1: PBS; G2: VNP; G3: VNP@PD-L1nb; G4: VNP@DOX-PBA; G5: VNP@PD-L1nb/DOX. Scale bar: 100 μm.

3. Conclusion

In this study, we designed a Salmonella typhimurium–based multifunctional platform (VNP@PD-L1nb/DOX) for chemoimmunotherapy in TNBC. The bacteria delivery platform sustainably secretes PD-L1nb and sensitively releases DOX into the tumor microenvironment. VNP@PD-L1nb/DOX significantly suppressed tumor growth in a murine TNBC model with induced ICD and promoted immune activation. This multifunctional platform enhanced intratumoral infiltration and activation of CD8+ T cells while concurrently depleting Treg cells. The synergy between ICD-driven immune priming and localized PD-L1 blockade culminated in a potent antitumor immune response. Collectively, our findings underscore the promise of engineered bacterial vectors as an integrated platform for chemoimmunotherapy in advanced solid tumors.

4. Experimental section

Chemicals and Reagents: The pBAD-PD-L1nb-His plasmid was synthesized by Genewiz (Suzhou, China). Yeast extract, tryptone, ampicillin, L-arabinose, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sangon Biotech (Shanghai). 4′,6-Diamidino-2-phenylindole (DAPI) and Cell Counting Kit-8 (CCK-8), BL21(DE3) competent cells, Dulbecco's Modified Eagle Medium (DMEM), and fetal bovine serum (FBS) were acquired from Beyotime (China). Anti-His primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-IgG (H + L) secondary antibody were obtained from Origene (USA). All antibodies used for blocking and flow cytometry in this study were purchased from BioLegend (USA). ELISA kits for serum tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were procured from KeyGEN (Jiangsu, China).

Cell Culture and Animals: The 4T1 murine breast cancer cell line and HEK-293T cells were obtained from Yeaden Biotechnology (Shanghai, China). All cells were cultured in DMEM supplemented with 10% FBS at 37 °C in a humidified incubator with 5% CO2. Female BALB/c mice were purchased from Hangzhou Hans Biotechnology Co., Ltd. (Hangzhou, China). All animal experiments were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Taizhou University (Zhejiang, China).

Preparation of VNP@PD-L1nb: The DNA sequence encoding SP-PD-L1nb-His was synthesized and cloned into the pBAD-Myc-His A vector (Genewiz, Suzhou, China). The resulting plasmid, pBAD-PD-L1nb-His, was transformed into VNP20009 competent cells. A single colony was inoculated into LB medium and cultured at 37 °C with shaking. When the OD600 reached 0.8, protein expression in VNP@PD-L1nb cells was induced by adding L-arabinose to a final concentration of 0.2% (w/v), followed by incubation at 37 °C for varying durations. Following induction, cultures were centrifuged to separate the supernatant and cell pellet. The presence of PD-L1nb protein in both fractions was analyzed by Western blot using an anti-His primary antibody.

Preparation of VNP@PD-L1nb/DOX: VNP@PD-L1nb/DOX was prepared by conjugating a phenylboronic acid–modified doxorubicin prodrug (DOX-PBA) to the surface of VNP@PD-L1nb. DOX-PBA was synthesized according to our previously reported procedures [41,42]. Briefly, VNP@PD-L1nb bacteria were collected and washed three times with phosphate-buffered saline (PBS). The bacterial suspension was then incubated with DOX-PBA at concentrations of 25, 50, 100, or 150 μg/mL for 4 h at room temperature. After incubation, unbound DOX-PBA was removed by repeated washing with PBS, yielding VNP@PD-L1nb/DOX. The amount of conjugated DOX-PBA was quantified by high-performance liquid chromatography (HPLC), and the drug-loading efficiency was calculated accordingly.

In Vitro PD-L1 Blocking Assay: Breast cancer cells 4T1 were seeded in 12-well plates at a density of 4 × 105 cells per well and cultured overnight. The next day, the medium was removed, and the cells were washed twice with PBS. Cells were treated with PBS control, purified PD-L1nb protein (100 μg/mL), or concentrated supernatant for 2 h at 37 °C. After washing with PBS, the treated cells were detached with trypsin. Cells were then incubated with PE-labeled-PD-L1 antibody (20 μg/mL diluted in 1% BSA) at 4 °C for 60 min. Cells were washed three times with PBS and analyzed by flow cytometry (gated on PE channel).

In vivo biodistribution analysis of VNP@PD-L1nb: All animal procedures were conducted in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Taizhou University (Zhejiang, China). To establish a triple-negative breast cancer (TNBC) mouse model, 1 × 106 4T1 cells were subcutaneously inoculated into the right mammary fat pad of 6-week-old female BALB/c mice. Once tumor volumes reached approximately 50 mm3, mice were randomly assigned to two groups: the BL21 control group and the VNP@PD-L1nb treatment group. Each mouse received an intravenous injection via the tail vein of 1 × 107 CFU of either BL21 or VNP@PD-L1nb bacteria engineered to express the luciferase gene. On day 7 post-injection, D-luciferin potassium salt was administered intravenously. Mice were then anesthetized with isoflurane and imaged using an in vivo imaging system (IVIS) to assess bacterial biodistribution. Following imaging, major organs and tumors were harvested ex vivo and photographed to evaluate bacterial accumulation across tissues.

Preparation of VNP@DOX-PBA and VNP@PD-L1nb/DOX: In this study, DOX-PBA was used to prepare DOX-coated bacteria. When the bacterial culture (VNP or VNP@PD-L1nb) reached an OD600 of 0.6, cells were harvested by centrifugation at 5000 rpm and resuspended in 1 mL of PBS. DOX-PBA was then added to the bacteria and incubated at room temperature for 4 h on a shaker (120 rpm). Unbounded DOX-PBA was removed by centrifugation, and the resulting bacterial pellet was resuspended in PBS.

Characteriazation of VNP@PD-L1nb/DOX: The conjugation of DOX-PBA to the engineered bacteria was evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry. Owing to the intrinsic fluorescence of DOX-PBA, VNP@PD-L1nb/DOX was visualized as a red fluorescent signal by CLSM. For flow cytometric analysis, 1 × 107 CFU of VNP@PD-L1nb or VNP@PD-L1nb/DOX were collected, washed with PBS, and analyzed using FITC channel. The fluorescence intensity of VNP@PD-L1nb/DOX was compared with that of unmodified VNP@PD-L1nb to assess the association of DOX-PBA with the bacterial cells.

In Vitro Drug Release: Drug release profile of VNP@PD-L1nb/DOX was evaluated using a dialysis method. For pH-responsive release, VNP@PD-L1nb/DOX were immersed in PBS buffers at pH 5.5 or 7.4 in dialysis bags (MW = 3500). For H2O2-responsive release, VNP@PD-L1nb/DOX was placed in PBS with H2O2 (100 μM) or without H2O2. Dialysis was performed at 37 °C with shaking at 180 rpm. At predetermined time points, 1 mL of the external solution was withdrawn and replaced with an equal volume of fresh buffer of the same pH. The concentration of released drug (DOX-PBA) was quantified using a UV-Vis spectrophotometer.

Cellular Uptake analysis: The internalization of VNP@DOX-PBA and VNP@PD-L1nb/DOX in 4T1 breast cancer cells was evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry. 4T1 cells were seeded in 24-well plates (1 × 104 cells/well) and cultured overnight in complete DMEM at 37 °C with 5% CO2. After 12 h of cell culture, cells were treated with DOX-PBA, VNP@DOX-PBA, or VNP@PD-L1nb/DOX for 4 h at 37 °C. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and counterstained with DAPI for 10 min. The cells were visualized using CLSM (Zeiss LSM710). For quantitative analysis of internalization, the treated cells were digested by trypsin and subjected to flow cytometry analysis (gated on PE channel).

Cytotoxicity Assay: The cytotoxic effects of VNP@DOX-PBA and VNP@PD-L1nb/DOX on breast cancer cells were evaluated using a CCK-8 assay. Briefly, 4T1 cells were seeded into 96-well plates at a density of 5 × 103 cells per well and allowed to adhere for 12 h. The cells were then treated with VNP@DOX-PBA or VNP@PD-L1nb/DOX at equivalent DOX concentrations. After 48 h of incubation, cell viability was determined using the CCK-8 assay according to the manufacturer's instructions. Absorbance was measured at 450 nm using a microplate reader.

In Vivo Antitumor Efficacy: All animal experiments complied with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Taizhou University (Taizhou, Zhejiang, China). The TNBC mouse model was established by subcutaneous injection of 1 × 106 4T1 cells into the right flank of BALB/c mice. When tumor volumes reached approximately 50 mm3, mice were randomly divided into five groups (n = 5): PBS, VNP, VNP@PD-L1nb, VNP@DOX-PBA (5 mg/kg DOX-PBA), or VNP@PD-L1nb/DOX (5 mg/kg DOX-PBA). Treatments were administered intravenously every 5 days for a total of three doses. Tumor volume was monitored using the formula: Volume = 0.5 × (length) × (width)2. Body weight and tumor dimensions were recorded at designated time points.

Flow Cytometry Analysis: Tumor tissues were minced into ∼1 mm3 fragments and digested in DMEM containing DNase I (0.25 mg/mL, Sigma-Aldrich) and collagenase IA (0.5 mg/mL, Sigma-Aldrich) at 37 °C for 30 min. The resulting cell suspension was filtered through a 200-mesh strainer and centrifuged at 1200 rpm for 5 min. Cells were stained with Zombie Violet Fixable Viability Kit and blocked with Fc receptor blocking antibody CD16/32 (clone 93). Surface and intracellular staining was performed using the following fluorophore-conjugated antibodies from BioLegend: CD45 (PerCP/Cyanine5.5; clone 30-F11), CD3 (APC; clone 17A2), CD8a (PE; clone 53-6.7), CD4 (FITC; clone GK1.5), Granzyme B (PE/Cyanine7; clone QA16A02), Perforin (APC; clone S16009A), CD25 (BV421; clone PC61), and Foxp3 (Alexa Fluor® 647; clone 150D). After three washes, samples were analyzed on a CytoFLEX flow cytometer (Beckman Coulter) using CytoExpert software. Tumor-infiltrating lymphocyte populations were identified as follows: CD8+ T cells (CD45+CD3+CD8+), Granzyme B+ CD8+ T cells (CD45+CD8+Granzyme B+), Perforin+ CD8+ T cells (CD45+CD8+Perforin+), and Tregs cells (CD45+CD3+CD4+Foxp3+).

Histological and Immunohistochemical Staining: Major organs (liver, spleen, kidney, heart, and lung) and tumors were harvested from mice receiving different treatments. For hematoxylin and eosin (H&E) staining, tissues were fixed, paraffin-embedded, sectioned, and stained. Tumor sections were also subjected to immunohistochemical analysis using Ki67 and calreticulin (CRT) antibodies. Apoptosis was detected using a colorimetric TUNEL Apoptosis Detection Kit (Beyotime, Shanghai). Additionally, CD8+ T cell infiltration was assessed by immunohistochemical staining with an PE-labeled anti-CD8 antibody.

Statistical analysis: All data are presented as mean ± standard deviation (SD). Statistical analyses were performed using SPSS software. One-way or two-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to determine statistical significance. The threshold of statistical significance was P < 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

CRediT authorship contribution statement

Longxue Guan: Data curation, Formal analysis, Investigation. Qinxiao Sun: Data curation, Formal analysis, Investigation. Hancheng Jiang: Data curation, Formal analysis. Dandan Liang: Data curation, Formal analysis. Shiqi Zhang: Investigation. Laili Huang: Investigation. Xingang Guan: Conceptualization, Funding acquisition, Supervision, Writing – original draft. Xiuli Hu: Conceptualization, Funding acquisition, Resources, Writing – review & editing. Guofu Chen: Funding acquisition.

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

L. Guan, Q. Sun, and H. Jiang contributed equally to this work. The authors sincerely acknowledge the financial support received from Zhejiang Provincial Natural Science Foundation of China (LY23C100001), Natural Science Foundation of Hebei Province (B2022202029) and the Government Guide the Development of Local Science and Technology Special Funds (226Z1203G).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mtbio.2026.103550.

Contributor Information

Xingang Guan, Email: guanxg@tzc.edu.cn.

Xiuli Hu, Email: huxiuli@hebut.edu.cn.

Guofu Chen, Email: wlcgf@163.com.

Appendix A. Supplementary data

The following is the supplementary data to this article:

Multimedia component 1
mmc1.docx (2.9MB, docx)

Data availability

Data will be made available on request.

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