Improving T cell expansion by optogenetically engineered bacteria-loaded MMP-2-responsive cyclophosphamide for antitumor immunotherapy

Abstract

The efficacy of antitumor immunotherapy is closely associated with the expansion of tumor-infiltrating CD8⁺ T cells. However, within the tumor microenvironment, CD8⁺ T cells often exhibit reduced proliferation due to persistent exposure to tumor antigens. The cytokine IL-2 is a potent growth factor that can drive the expansion of tumor-infiltrating lymphocytes. While its clinical application has been severely limited by systemic toxicity and in vivo instability. To address these challenges, we have developed a dual-responsive system (EcN_IL-2@UCNP/Gel-CTX) leveraging the hypoxic tropisms of E. coli Nissle 1917(EcN). This system is capable of producing IL-2 in situ upon near-infrared (NIR) irradiation and releasing low-dose cyclophosphamide (CTX) in response to matrix metalloproteinase-2 (MMP-2) in the tumor microenvironment. The EcN_IL-2@UCNP/Gel-CTX system not only drives the expansion of CD8⁺ T cells and boost the activity of NK cells but also reduces Treg cell populations, thereby remodeling the immune microenvironment and eliciting robust tumor-specific immune responses in H22 subcutaneous tumors in mice and confers long-term protection against tumor rechallenge by promoting the generation of durable memory T cells. Our findings provide an both light and tumor microenvironment responsive platform for enhanced cancer immunotherapy.

Graphical Abstract

We have developed a dual-responsive system, EcN_IL-2@UCNP/Gel-CTX, which enables in situ interleukin-2 (IL-2) production under near-infrared (NIR) irradiation and releases low-dose cyclophosphamide (CTX) in response to matrix metalloproteinase-2 (MMP-2) within the tumor microenvironment. This system not only enhances CD8⁺ T cell expansion and NK cell activity but also suppresses regulatory T cell (Treg) populations, effectively remodeling the immunosuppressive tumor microenvironment. In murine H22 subcutaneous tumor models, EcN_IL-2@UCNP/Gel-CTX elicited potent tumor-specific immune responses and conferred long-term protection against tumor rechallenge by inducing durable memory T cell generation.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-025-03801-4.

Introduction

Immune checkpoint therapy relies on the activation of cytotoxic T lymphocytes that tumors exploit to evade immune destruction, and has shown notable therapeutic benefit in clinical cancer management [1]. However, its clinical benefits are only observed in certain patient subsets and limited to specific tumor types [2, 3]. Studies indicate that tumor infiltrating CD8⁺ T cells often exhibit decreased proliferation and cytotoxic capability due to continuously stimulation of tumor antigens and inflammatory factors within solid tumors [4]. Moreover, many inhibitory cells in the tumor microenvironment, such as, regulatory T cells (Tregs), release immunosuppressive cytokines to further impair and inhibit the anti-tumor functions of CD8⁺ T cells [5]. These multiple resistance mechanisms highlight the pressing need for effective combinational strategies that target these resistance pathways and improve clinical responses.

IL-2 is tightly correlated to T cell proliferation and effector functions, such as potentiating the expansion of CD8⁺ T cells [6]. IL-2 was approved by the US Food and Drug Administration for the treatments of melanoma and renal cell carcinoma. However, its clinical application is limited by significant systemic toxicity and the short half-life time [7, 8]. This limitation highlights the need for advanced delivery systems that increase IL-2 concentrations specifically at tumor sites, minimizing toxicity, dosing frequency, and improving CD8⁺ T cell proliferation [9]. Although nanovectors had increased the targeted delivery of IL-2 to some extent, physical and physiological barriers within the body limit its delivery efficiency [10].

Certain anaerobic or facultative anaerobic bacteria present a pronounced hypoxia tropism, enabling the selective accumulation and colonization within both solid and metastatic tumors [11]. A variety of bacteria, including Salmonella, Escherichia coli (E. coli), Clostridium, and Bifidobacterium have been developed for tumor treatments [12]. Attenuated bacteria hold innate immunomodulatory effect, activating immune cells and promoting cytokine release [13]. In addition, these bacteria can be conveniently modified by genetic or chemical methods to serve as tailored drug-delivery vehicles [14]. With development of synthetic biology, E. coli has been engineered as a programmable living vector for producing cytokines, immunomodulators, or chemotherapeutics in situ, thereby improving both safety and therapeutic efficacy [15]. The production of IL-2 by genetically-engineered tumor-targeted bacteria may largely decrease the systemic toxicity of IL-2 [16]. Moreover, the production of IL-2 by engineered bacteria within the tumor may overcome its short half-life during circulation [17]. Therefore, localized production of IL-2 brings a promising strategy for driving CD8⁺ T cell proliferation for tumor cell killing.

Cyclophosphamide (CTX), a chemotherapeutic drug, has been included in various regimens for its immunomodulatory effects [18–20]. Low-dose CTX (≤ 20 mg/kg) selectively reduces Treg number and function while enhancing CD8⁺ T and NK cell activity [21–24]. IL-2 activates effector T cells but also expands CD25⁺ Tregs, attenuating antitumor immunity [25, 26]. The combination of IL-2 with low-dose CTX elicited synergistic effects, amplifying IL-2-mediated immune activation [27–30]. Preclinical and clinical studies show that CTX prevents IL-2–induced Treg expansion, remodels their function, and improves the immune milieu, thereby amplifying IL-2-mediated antitumor responses [31, 32]. However, the “on-demand” production of IL-2 and release of CTX in situ remained a challenge. Herein, a both light and matrix metalloproteinase-2 (MMP-2) response system (EcN_IL−2@UCNP/Gel-CTX) was prepared, producing IL-2 upon near-infrared (NIR) light irradiation, and release of low-dose cyclophosphamide (CTX) responsive to MMP-2. A blue light-sensitive system for IL-2 expression by integrating the EL222 sensor gene and the IL-2-mCherry fusion gene were transferred into the pET20b plasmid (pET20b-EL222-IL-2-mCherry). The mCherry protein serves as a reporter, enabling the construction of an IL-2 expression system in E. coli. Due to the limited tissue penetration of blue light, we synthesized upconversion nanoparticles (UCNPs) that convert NIR light to blue light. Additionally, gelatin nanoparticles loaded with low-dose CTX, responding to MMP-2, were synthesized using a one-phase condensation method. By employing EDC/NHS amide coupling, UCNPs and Gel-CTX were conjugated onto EcN_IL−2, creating a drug-loaded engineered bacterial system (EcN_IL−2@UCNP/Gel-CTX). This system allows for NIR-induced in situ expression of IL-2, alongside the MMP-2 responsive release of CTX. Strong antitumor immune responses were induced in vitro and in vivo (Fig. 1).

Fig. 1.

Optogenetics-engineered EcN _IL−2 conjugation with MMP-2 responding Gel-CTX (EcN_IL−2@UCNP/Gel-CTX) for tumor specific immunotherapy. (A) The preparation of EcN_IL−2@UCNP/Gel-CTX. (B) Mechanism for production of secretion of IL-2 directly from EcN_IL−2@UCNP/Gel-CTX upon 808 nm irradiation, and MMP-2 simultaneous release of CTX. EcN_IL−2@UCNP/Gel-CTX activates CTLs, and inhibits Treg cells for enhancing tumor immunotherapy

Results and discussion

Preparation and characterization of EcN_IL−2@UCNP/Gel-CTX

In this study, the recombinant plasmid pET20b-EL222-IL-2/mCherry was constructed, in which blue light-sensitive EL222 and IL-2 gene sequences were inserted into plasmid pET20b, and mCherry was used as a reporter gene (Fig. S1). The recombinant plasmid was transfected into EcN to obtain EcN_IL−2 with the ability of expressing the secretory IL-2 under blue light-irradiation. Due to the shallow penetration of blue light, UCNPs with the ability to convert deep-penetrating NIR into blue light were conjugated onto the surface of EcN_IL−2. At the same time, Gel loaded with CTX, was coupled with EcN_IL−2 via EDC/NHS chemistry to construct an EcN_IL−2@UCNP/Gel-CTX with both light and MMP-2 response abilities.

TEM results showed that UCNPs were dispersed uniformly in hexagonal form (Fig. 2A). As shown in Fig. 2B and C, the size of UCNP was 66.91 ± 1.87 nm and the zeta potential was 24.53 ± 0.45 mV. The size of spherical Gel-CTX particle was 207.67 ± 4.61 nm, and zeta potential was − 3.39 ± 0.57 mV by DLS. After coupling UCNP/Gel-CTX with EcN_IL−2, the size of EcN_IL−2@UCNP/Gel-CTX was 1827.67 ± 127.77 nm, and zeta potential was − 1.13 ± 0.07 mV. Under NIR irradiation, the blue light emission from the EcN_IL−2@UCNP and EcN_IL−2@UCNP/Gel-CTX were similar to that from the UCNPs (Fig. 2D). While no blue light emission was found in the EcN_IL−2 and Gel-CTX groups. These results indicated that UCNPs were successfully conjugated with bacteria and the conjugation does not affect the light conversion ability of UCNPs.

Fig. 2.

Characterizations of EcNIL-2@UCNP/Gel-CTX. (A) TEM images of EcN_IL−2, UCNPs, Gel-CTX, EcN_IL−2@UCNP, and EcN_IL−2@UCNP/Gel-CTX. Scale bar: 200 nm in UCNP and Gel-CTX columns, and 500 nm in others. (B) Particle size distribution and (C) Zeta potential of UCNPs, Gel-CTX, EcN_IL−2, EcN_IL−2@UCNP, EcN_IL−2@Gel-CTX, and EcN_IL−2@UCNP/Gel-CTX measured by DLS (n = 3). (D) Emission spectra of UCNPs, Gel-CTX, EcN_IL−2, EcN_IL−2@UCNP, EcN_IL−2@Gel-CTX, and EcN_IL−2@UCNP/Gel-CTX. (E) The colocalization of bacteria and gelatin nanoparticles in the EcN_IL−2@UCNP/Gel-CTX under confocal microscopy. Bacterial membranes were stained with DiI (red) and gelatin nanoparticles were stained by loading FITC (green). Scale bar: 20 μm. (F) Drug release from Gel-CTX with or without adding MMP-2. (G) The mCherry intensity of EcN_IL−2@UCNP/Gel-CTX by analysis of flow cytometry after near-infrared light irradiation 3 cycles. (H) Confocal images of EcN_IL−2, EcN_IL−2@UCNP, and EcN_IL−2@UCNP/Gel-CTX after NIR light illumination (1 W/cm², 30 min). Scale bar: 10 μm. (I) IL-2 expression in the bacteria and supernatant fractions of EcN_IL−2, EcN_IL−2@UCNP, and EcN_IL−2@UCNP/Gel-CTX with or without light exposure, detected by ELISA. G1: UCNPs, G2: Gel-CTX, G3: EcN_IL−2, G4: EcN_IL−2@UCNP, G5: EcN_IL−2@Gel-CTX, G6: EcN_IL−2@UCNP/Gel-CTX. All values are expressed as mean ± s. e. m. Statistical analysis was performed using one-way ANOVA with Tukey’s post-test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, ns indicates no significant difference

To detect the co-location of EcN_IL−2 and CTX, CTX in Gel was replaced by FITC and the membrane of EcN_IL−2 was stained with DiI. The images under laser confocal microscopy showed Gel-FITC attached onto the bacteria (Fig. 2E). CTX drug loading rate was 5.06% in Gel using HPLC method, which indicated that the prepared nanoparticles have high drug loading efficiency. As shown in the Figure S2, the particle size remained unchanged across all media, demonstrating excellent dimensional stability in both PBS and serum-containing environments. According to the high expression of MMP-2 in the tumor microenvironment, 10 µg/mL MMP-2 were added into Gel-CTX solution. Thereafter, the release behavior of CTX in Gel-CTX nanoparticles was detected by HPLC, and the release curves of CTX with or without MMP-2 were drawn, respectively (Fig. 2F). CTX released from Gel-CTX achieves 70% in 24 h after treatment with 10 µg/mL MMP-2, indicating that Gel-CTX exhibited good sustained-release properties.

As shown in the growth curve (Figure S3), all three groups (EcN_IL−2, EcN_IL−2@UCNP, and EcN_IL−2@UCNP/Gel-CTX) exhibited a typical bacterial growth trend, including an initial lag phase (0–2 h), onset of logarithmic growth (4–6 h), and rapid exponential increase at 8 h. Importantly, the engineered strains maintained growth kinetics comparable to wild-type EcN, indicating that genetic modification and nanoparticle conjugation had no significant effect on the growth rate of EcN_IL−2.

The fluorescence signal of mCherry could be detected after blue light illumination (BL+), and increased in a time-dependent manner by analysis of flow cytometry, which demonstrated that the blue-light responsive EcN_IL−2 was successfully constructed (Fig. S4). Moreover, in Fig. S5A, the fluorescence signal of mCherry in the EcN_IL−2 (BL+) group was higher than that in the EcN_IL−2@UCNP (NIR+) group, which may be attributed to the partial loss of energy during light conversion. Furthermore, western blot results showed that the expression of mCherry both in bacteria and their supernatants in the EcN_IL−2 (BL+) and EcN_IL−2@UCNP (NIR+) groups (Fig. S5B). Altogether, after coupling with UCNP, the EcN_IL−2 can express and secrete IL-2 upon NIR irradiation. After 30 min of NIR irradiation, the fluorescence intensity of mCherry significantly increased (Fig. S6). Moreover, the fluorescence intensity of mCherry in the EcN_IL−2@UCNP/Gel-CTX group increased in a time and power-dependent manner, which indicates that IL-2 expression can be controlled precisely (Fig. 2G and S7). The images under the confocal microscope showed the similar results (Fig. 2H). The IL-2 levels in the supernatant of EcN_IL−2@UCNP/Gel-CTX (NIR+) reached 718.08 pg/mL, as determined by ELISA (Fig. 2I). These results indicate that EcN_IL−2@UCNP/Gel-CTX has super capability to induce the expression of IL-2 under NIR.

Immunoactivation of EcN_IL−2@UCNP/Gel-CTX in vitro

Tumor infiltrating lymphocytes (TILs) are tightly associated with the efficiency of tumor immunotherapy [33, 34]. While IL-2 played a crucial role in the expansion of TILs, especially in T cells and NK cells. In this study, TILs were separated from tumors, and incubated with EcN_IL−2@UCNP/Gel-CTX under NIR irradiation and MMP-2 (10 µg/mL) to analyze the activation of immune cells.

The activation of T cells requires both the engagement of the T cell receptor (TCR) with antigens presented by antigen-presenting cells (APCs) and the interaction of the co-stimulatory receptor CD28 on T cells with the co-stimulatory ligands B7-1/B7-2 on APCs [35]. Tumor-infiltrating lymphocytes (TILs) are typically stimulated by antigen-presenting cells (APCs) in the tumor microenvironment. Therefore, TILs isolated from a cancer biopsy can be greatly expanded by adding IL-2, bypassing the interaction with APCs [36]. In this study, TILs collected from tumors were directly incubated with the supernatant of EcN_IL−2@UCNP/Gel-CTX after NIR irradiation and MMP-2 digestion. As shown in Fig. 3A, the number of CD8⁺ T cells increased significantly in the EcN_IL−2@UCNP (NIR+) and EcN_IL−2@UCNP/Gel-CTX (NIR+, MMP- 2+) groups. Among them, CD8⁺ T cells in the EcN_IL−2@UCNP/Gel-CTX (NIR+, MMP-2+) group are higher than other groups, presenting 2.07 and 1.73-fold higher than PBS and EcN_IL−2 groups, respectively (Fig. 3B). Altogether, the combination of NIR-induced IL-2 and low dose CTX can activate T cells significantly. Moreover, NK cells (CD49b⁺) activation in the EcN_IL−2@UCNP/Gel-CTX (NIR+, MMP-2+) group presented the similar trends to that of T cells (Fig. 3C and D). IL-2 is generally considered promoting T-cell proliferation and enhancing effector T-cell function, yet unexpectedly inducing immune tolerance and promoting Tregs development. As shown in Fig. 3E and F, the ratio of Tregs increased in the EcN_IL−2@UCNP (NIR+) group. While Tregs (CD25⁺/Foxp3⁺) were largely inhibited by the supernatant of EcN_IL−2@UCNP/Gel-CTX due to low dose CTX release after MMP-2 digestion.

Fig. 3.

EcN_IL−2@UCNP/Gel-CTX activates immune response in vitro. (A) Representative flow cytometry plots and (B) statistical results showing CD8⁺ T cells activation in TILs after different treatments. (C) Representative flow cytometry plots and (D) statistical results showing NK cells activation after different treatments. (E) Representative flow cytometry plots and (F) statistical results showing the effect of different treatments on Tregs in TILs. (G) IFN-γ and (H) Gra B expression levels in TILs after different treatments, detected by ELISA. (I) The killing effect of TILs stimulated by different treatments on tumor cells in vitro. G1: PBS, G2: CTX, G3: EcN_IL−2, G4: EcN_IL−2 (NIR+), G5: EcN_IL−2@UCNP, G6: EcN_IL−2@UCNP (NIR+), G7: EcN_IL−2@UCNP/Gel-CTX (MMP-2+), G8: EcN_IL−2@UCNP/Gel-CTX (NIR+, MMP-2+)

IFN-γ and Granzyme B (Gra B) were the key markers of active T cells and NK cells. IFN-γ in the EcN_IL−2@UCNP/Gel-CTX (NIR+, MMP-2+) group was 2.45, 1,85 and 1.24-fold higher than that in the PBS, EcN_IL−2 and EcN_IL−2@UCNP (NIR+) groups, respectively (Fig. 3G). The level of Gra B in the EcN_IL−2@UCNP/Gel-CTX (NIR+, MMP-2+) group was 2.71, 1.69 and 1.37-fold higher than the PBS, EcN_IL−2 and EcN_IL−2@UCNP (NIR+) groups, respectively (Fig. 3H). Altogether, NIR-inducing IL-2 combined with low dose CTX can effectively activate TILs, and promote TILs to secrete antitumor elements.

To exclude the direct anti-tumor effect, EcN_IL−2@UCNP/Gel-CTX were co-cultured with H22 cells in vitro. No significant changes in cell viability were found (Fig. S8). Afterwards, to mimic the immune cells killing tumor cells in vivo, TILs after different treatments were co-incubated with H22 cells. As shown in the Fig. 3I, the cytotoxicity in the EcN_IL−2@UCNP/Gel-CTX (NIR+, MMP-2+) group achieved to 21.77% higher than 15.93% in the EcN_IL−2@UCNP (NIR+) group. The result provided evidence that the ability of TILs to kill tumor cells was significantly enhanced in the EcN_IL−2@UCNP/Gel-CTX (NIR+, MMP-2+) group.

The biodistribution of EcN_IL−2@UCNP/Gel-CTX in tumor-bearing mice

As shown in Fig. 4A, the bio-distribution of EcN_IL−2@UCNP/Gel-CTX in vivo was detected by IVIS imaging system after staining with IR-780. At different time points, the fluorescence signal increased over time, and achieved to the peak at 48 h (Fig. 4B). Moreover, the major organs, such as heart, liver, spleen, lung, kidney, and tumor were resected for analysis of fluorescence signal at 72 h. The results showed that the fluorescence intensity in tumors was far higher than that in other tissues (Fig. 4C and D). After intravenous administration of EcN_IL−2@UCNP/Gel-CTX (Fig. S9), blood CFUs declined sharply within the first 12 h and became nearly undetectable within 24–72 h. Low levels of colonies were detected in normal organs such as the heart, liver, spleen, lung, and kidney within 72 h. In contrast, tumor tissues exhibited markedly higher bacterial accumulation. These findings demonstrate the tumor-targeting ability of EcN_IL−2@UCNP/Gel-CTX.

Fig. 4.

Antitumor efficacy of EcN_IL−2@UCNP/Gel-CTX in vivo. (A) Fluorescence images H22 tumor-bearing mice after tail vein injection of IR780-labeled EcN_IL−2@UCNP/Gel-CTX at different timepoints. (B) Quantitative analysis of the fluorescence images over time (n = 3). (C) Fluorescence images of major organs and tumors collected at 72 h post-injection (D) Quantitative analysis of the fluorescence in the major organs and tumors. (E) Schematic of the treatment regimen in H22 tumor -bearing mice. (F) Tumor growth curves and (G) tumor images after different treatments. (H) Tumor weights of different groups. (I) H&E, TUNEL, and Ki67 staining of tumor tissues from different treatment groups. Scale bar: 50 μm. G1: PBS, G2: CTX, G3: EcN_IL−2, G4: EcN_IL−2 (NIR+), G5: EcN_IL−2@UCNP, G6: EcN_IL−2@UCNP (NIR+), G7: EcN_IL−2@UCNP/Gel-CTX, G8: EcN_IL−2@UCNP/Gel-CTX (NIR+)

In vivo antitumor efficacy of EcN_IL−2@UCNP/Gel-CTX (NIR+)

The antitumor effects of EcN_IL−2@UCNP/Gel-CTX (NIR+) were further evaluated in vivo. Eight groups were divided as follows: PBS, CTX, EcN_IL−2, EcN_IL−2 (NIR+), EcN_IL−2@UCNP, EcN_IL−2@UCNP (NIR+), EcN_IL−2@UCNP/Gel-CTX, and EcN_IL−2@UCNP/Gel-CTX (NIR+). The experiment design is shown in Fig. 4E, no significant difference was found between PBS and CTX groups, which indicated low dose CTX had no significant anti-tumor efficacy. Tumors were inhibited in EcN_IL−2, EcN_IL−2 (NIR+), EcN_IL−2@UCNP and EcN_IL−2@UCNP/Gel-CTX to some extent, which may be attributed to the poor immunogenicity of EcN. Tumor suppression was achieved at 62.20% and 85.05% in EcN_IL−2@UCNP (NIR+) and EcN_IL−2@UCNP/Gel-CTX (NIR+) groups, respectively (Fig. 4G). Both tumor images and tumor weight data showed consistent results (Fig. 4F and H). The results indicated that IL-2 combined with low-dose CTX could inhibit the growth of H22 tumors. After staining by H&E, TUNEL and Ki67 staining, the tumor tissues in the EcN_IL−2@UCNP/Gel-CTX (NIR+) group showed the weakest cell proliferation (Fig. 4I).

In vivo anti-tumor immune activation of EcN_IL−2@UCNP/Gel-CTX (NIR+)

To further investigate the antitumor mechanism of EcN_IL−2@UCNP/Gel-CTX (NIR+), DCs maturation in lymph nodes was analyzed by flow cytometry after treatments on the 3rd day after the last irradiation (after second i.v. injection). As shown in Fig. 5A and B, CTX alone cannot induce DCs maturation, which is similar to the PBS group. DCs maturation was found to some extent in the EcN_IL−2 (NIR+), EcN_IL−2, EcN_IL−2@UCNP, and EcN_IL−2@UCNP/Gel-CTX groups. CD80⁺ CD86⁺ DCs ratio in EcN_IL−2@UCNP (NIR+) and EcN_IL−2@UCNP/Gel-CTX (NIR+) groups was 1.74 and 1.84-fold higher than PBS group, respectively. CD8⁺ T cells in tumor-draining lymph nodes (TDLN) in EcN_IL−2@UCNP (NIR+) and EcN_IL−2@UCNP/Gel-CTX (NIR+) groups achieved at 42.8% and 43.9%, respectively, higher than other groups (Fig. 5C). The highest ratio of CD8⁺/CD4⁺ T cells was found in the EcN_IL−2@UCNP/Gel-CTX (NIR+) group (Fig. S10).

Fig. 5.

In vivo immune activation effects of EcN_IL−2@UCNP/Gel-CTX (NIR+). (A) Flow cytometry analysis of DCs maturation (CD11c⁺ CD80⁺ CD86⁺) in TDLN from different groups. (B) Quantification of mature DCs. (C) Proportion of CD8⁺ T cells in TDLN. (D) Flow cytometry analysis of T cells in the tumors after different treatments. (E) Proportion of CD8⁺ T cells and (F) NK cells in tumors. (G)Immunofluorescence staining of Tregs (Foxp3⁺ T cells), IFN-γ, and Gra B in tumor tissues. Scale bar: 50 μm. (H) Flow cytometry analysis of T cells in the spleen of mice after different treatments. (I) Quantification of CD8⁺ T cells and (J) CD8⁺/CD4⁺T ratio in spleen. (K) The serum levels of IFN-γ, (L) TNF-α, (M) IL-10, and (N) TGF-β in different groups by ELISA. G1: PBS, G2: CTX, G3: EcN_IL−2, G4: EcN_IL−2 (NIR+), G5: EcN_IL−2@UCNP, G6: EcN_IL−2@UCNP (NIR+), G7: EcN_IL−2@UCNP/Gel-CTX, G8: EcN_IL−2@UCNP/Gel-CTX (NIR+)

To evaluate the immune response induced by EcN_IL−2@UCNP/Gel-CTX (NIR+) in H22 tumor-bearing mice, CD8⁺ T and CD4⁺ T cells in tumors were analyzed by flow cytometry on the 5th day after the last irradiation. As shown in Fig. 5D and E, there was no significant difference in the infiltration of CD8⁺ T cells among the EcN_IL−2, EcN_IL−2 (NIR+), EcN_IL−2@UCNP, and EcN_IL−2@UCNP/Gel-CTX groups, while all showed slightly higher levels compared to both the PBS and CTX groups. The highest ratio of CD8⁺ T cells was presented in the EcN_IL−2@UCNP/Gel-CTX (NIR+) group, which was 2.73, 1.73 and 1.29-fold higher than that in the PBS, EcN_IL−2, and EcN_IL−2@UCNP (NIR+) groups, respectively. A similar tendency was found in CD4⁺ T cells in deferent groups (Fig. S11). The ratio of NK cells in tumor tissues in the EcN_IL−2@UCNP (NIR+) group was also higher than that in other groups (Fig. 5F, S12). Altogether, the combination of IL-2 and low dose CTX greatly promoted the infiltration and activation of T cells and NK cells.

Moreover, fluorescence intensity of Foxp3, a marker of Tregs, increased in the EcN_IL−2@UCNP (NIR+) group, whereas it largely decreased in the EcN_IL−2@UCNP/Gel-CTX (NIR+) group after immune-fluorescence staining, which may be attributed to the fact that low dose CTX inhibit Tregs induced by IL-2 (Fig. 5G). Fluorescence intensity of Granz B and IFN-γ in the EcN_IL−2@UCNP/Gel-CTX (NIR+) group was higher than that in other groups, which was consistent with the results in vitro.

To explore the systemic immune activation mediated by EcN_IL−2@UCNP/Gel-CTX (NIR+) in H22 tumor-bearing mice, spleen tissues were harvested and homogenized to obtain single-cell suspensions for analysis. The proportions of CD8⁺T cells and CD4⁺T cells were detected by flow cytometry. The test results are shown in Fig. 5H, after treatment with EcN_IL−2@UCNP/Gel-CTX (NIR+), the proportion of CD8⁺ T cells in splenic T cells was 15.6%, which was significantly higher than the 8.33% in the PBS group (Fig. 5I). The ratio of CD8⁺ T to CD4⁺ T cells in the EcN_IL−2@UCNP/Gel-CTX (NIR+) group was higher than other groups (Fig. 5J). The results indicated that EcN_IL−2@UCNP/Gel-CTX (NIR+) treatment can significantly increase the infiltration of CD8⁺T cells in the spleen of mice, causing a systemic immune response.

Moreover, the level of IFN-γ in the EcN_IL−2@UCNP/Gel-CTX (NIR+) group, was 2.92 and 2.65-fold higher than that in PBS and CTX groups, respectively (Fig. 5K). And TNF-α was 2.07 and 1.96-fold higher than that in PBS and CTX groups, respectively (Fig. 5L). Treg-related cytokines IL-10 and TGF-β in the EcN_IL−2@UCNP/Gel-CTX (NIR+) group decreased by 1.15 and 1.01-fold compared to that in the EcN_IL−2@UCNP (NIR+) group (Fig. 5M and N). Altogether, EcN_IL−2@UCNP/Gel-CTX (NIR+) activated the immune response in TME and improved the tumor immune suppression microenvironments.

Long-term immune memory

To investigate the immune memory induced by EcN_IL−2@UCNP/Gel-CTX (NIR+), experiments were designed as shown in Fig. 6A. T cells in spleen were analyzed on the 7th day after the last treatment in part of the mice. Tumors were resected on the 8th day after the last treatment in the other tumor-bearing mice. On the 18th day after the last treatment, H22 cells were rechallenged subcutaneously on the opposite side. As shown in Fig. S13, the significant antitumor effect of EcN_IL−2@UCNP Gel-CTX (NIR+) was found after three administrations. Tumor inhibition achieved to 92.2% after administration of EcN_IL−2@UCNP/Gel-CTX (NIR+) 3 times.

Fig. 6.

Long-term immune memory induced by EcN_IL−2@UCNP/Gel-CTX (NIR+). (A) Schematic diagram of the immune memory experiment design. (B) Flow cytometry analysis of the proportion of effector memory T cells in the spleen of H22 tumor-bearing mice in different groups and (C) quantitative analysis. (D) Specific killing in different groups. (E) Tumor image, (F) tumor growth curve and (G) and tumor weight in the PBS and EcN_IL−2@UCNP/Gel-CTX (NIR+) groups after tumor rechallenge. (H) Images and (I) quantitative analysis of spleens, and (J) Images and (K) quantitative analysis of lymph nodes from control and treatment groups. G1: PBS, G2: CTX, G3: EcN_IL−2, G4: EcN_IL−2 (NIR+), G5: EcN_IL−2@UCNP, G6: EcN_IL−2@UCNP (NIR+), G7: EcN_IL−2@UCNP/Gel-CTX, G8: EcN_IL−2@UCNP/Gel-CTX (NIR+)

As shown in Fig. 6B and C, T memory cells in spleen could be induced by EcN_IL−2@UCNP (NIR+) and EcN_IL−2@UCNP/Gel-CTX (NIR+). The highest immune memory was found in the EcN_IL−2@UCNP/Gel-CTX (NIR+) group. Moreover, lymphocytes collected from the spleen were treated with H22 tumor antigen from ultrasound-induced cell lysis, showing significant specific H22 cells killing effects, which indicates EcN_IL−2@UCNP/Gel-CTX (NIR+) could effectively train the immune memory (Fig. 6D).

Furthermore, the tumor rechallenge result showed that tumor growth was completely inhibited in the EcN_IL−2@UCNP/Gel-CTX (NIR+) group. Altogether, long-term immune memory could be induced by EcN_IL−2@UCNP/Gel-CTX (NIR+) to inhibit tumor cell rechallenge (Fig. 6E, F and G). This suggests that EcN_IL-2@UCNP/Gel-CTX (NIR+) treatment induced robust immunological memory, enabling a rapid and effective immune response upon tumor antigen re-exposure, thereby preventing tumor recurrence.

As shown in Fig. 6H and K, the size and weight of spleens and lymph nodes in the EcN_IL−2@UCNP/Gel-CTX (NIR+) group were lower than that in the control group, which indicated that the inflammation was not overstimulated in the EcN_IL−2@UCNP/Gel-CTX (NIR+) group.

Biosafety analysis

Biochemical analysis was carried out to detect alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactic dehydrogenase (LDH), creatine kinase (CK), urea nitrogen (BUN), and creatinine (CREA) in sera for evaluating systemic safety of EcN_IL−2@UCNP/Gel-CTX(NIR+). As shown in Fig. 7A-F, those blood biochemical parameters were in the normal range. Mouse body weight (Fig. S14) maintained stability during the monitoring period, and the staining of different tissues by H&E (Fig. 7G) showed no significant changes after treatment with EcN_IL−2@UCNP/Gel-CTX (NIR+). Therefore, EcN_IL−2@UCNP/Gel-CTX (NIR+) presented good biocompatibility in vivo.

Fig. 7.

Biosafety assessment of EcN_IL-2@UCNP/Gel-CTX (NIR+) treatment. (A) Serum levels of ALT, (B) AST, (C) LDH, (D) CK, (E) BUN, and (F) CREA. (G) H&E staining of heart, liver, spleen, lung, and kidney tissue sections from H22 tumor-bearing mice after different treatments. Scale bar: 50 μm. G1: PBS, G2: CTX, G3: EcN_IL-2, G4: EcN_IL-2 (NIR+), G5: EcN_IL-2@UCNP, G6: EcN_IL-2@UCNP (NIR+), G7: EcN_IL-2@UCNP/Gel-CTX, G8: EcN_IL-2@UCNP/Gel-CTX (NIR+).

Conclusion

Tumor-infiltrating CD8⁺ T cells, which are closely associated with the efficacy of antitumor immunotherapy, are often insufficient for effectively killing tumor cells within the tumor microenvironment. The cytokine IL-2 can drive the expansion of activated T populations. However, its clinical application is limited by systemic toxicity and in vivo instability. To address these limitations, we constructed a genetically engineered bacteria-based system (EcN_IL−2@UCNP/Gel-CTX) that responds to near-infrared (NIR) light and matrix metalloproteinase-2 (MMP-2) for cancer immunotherapy. Specifically, IL-2 is secreted upon exposure to NIR light, while the release of cyclophosphamide (CTX) is triggered by MMP-2 from the EcN_IL−2@UCNP/Gel-CTX system. This dual-responsive design expands CD8⁺ T cells, and NK cells within the tumor while inhibiting Tregs, thereby eliciting robust anti-tumor immune responses and long-lasting immune memory in mice. Compared with previously reported CTX/IL-2 combination therapies, the EcN_IL−2@UCNP/Gel-CTX platform offers several advantages. Foremost is its dual-responsive controlled-release mechanism, which enables spatiotemporal coordination of CTX and IL-2 delivery within the immunosuppressive TME. By localizing IL-2 release to sites of TME while restricting CTX activation to the core of solid tumors, EcN_IL−2@UCNP/Gel-CTX minimizes peripheral exposure, thereby widening the therapeutic index. Nevertheless, although up-conversion nanoparticles provide deep penetration of NIR light, their long-term biocompatibility remains debated, necessitating further optimization in the follow-up studies.

Methods

Bacteria and plasmid

Escherichia. coli Nissle1917 (EcN) was used in this study. The IL-2 sequences incorporated in the plasmid construct is murine IL-2 (Mus musculus), which was selected to match the immune system of BALB/c mice used in the study. The recombinant plasmid (pET20b-EL222-IL-2-mCherry) was constructed by Wuhan Miaoling Biotechnology Co. Ltd, and was transfected into EcN by electroporation (2.5 kV, 25 µF, 200 Ω, 5 ms). Plasmid-transfected EcN (EcN_IL−2) was cultured in Luria-Bertani (LB) agar plates or LB medium supplemented with antibiotics (100 µg∙mL^− 1 ampicillin). The engineered bacteria were cultured in a shaking incubator (180 rpm) at 37 °C. The detailed plasmid information was shown in Fig. S1.

Synthesis and surface modification of NaYF4:Yb, Tm@NaYbF4,Nd@NaGdF4 (UCNPs)

The synthesis and modification of UCNPs in this study were performed according to the previous reports [37, 38].

Preparation of Gel-CTX

Type A gelatin (0.1 g) and CTX (8 mg) were dissolved in 10 mL of deionized water at 45℃ under stirring to ensure full swelling. Subsequently, 150 µL of Tween-20 were added under stirring. The pH was adjusted to 7.0 using a 0.5 M NaOH. Afterwards, approximately 7 mL of Na₂SO₄ solution (0.5 M) was added dropwise to achieve a continuous turbidity. Then 1 mL of isopropanol was added to obtain the clear solution. Thereafter, 25% glutaraldehyde was added into the mixture by a constant flow injection pump at a rate of 0.05 mL/min under the stirring speed of 800 rpm for 15 min. The unreacted glutaraldehyde and the rest of the unloaded drugs were removed using an ultrafiltration centrifugation method. The prepared gelatin nanoparticles were then lyophilized for storage.

Drug release and stability profile of Gel-CTX

The content of free CTX was determined using HPLC with an Elite C18 column, and a detection wavelength was set at 197.0 nm. The mobile phase consisted of a mixture of acetonitrile and water at a ratio of 30:70. The flow rate was maintained at 1.2 mL/min.

Preparation of EcN_IL−2@UCNP and EcN_IL−2@UCNP/Gel-CTX

UCNPs (40 mg), Gel-CTX (40 mg), EDC (27 mg) and sulfo-NHS (14 mg) were added into MES buffer (10 mL, 0.1 M, pH 6.0), and then stirred at room temperature for 4 h. After washing with PBS for 3 times, 10⁹ CFU EcN_IL−2 in PBS were added into the deposition, and stirred at room temperature for 12 h. EcN_IL−2@UCNP/Gel-CTX were obtained after removing the remaining UCNPs and Gel-CTX by centrifugation (8000 rpm, 10 min). EcN_IL−2@UCNP/Gel-CTX were resuspended in PBS or LB culture medium. EcN_IL−2@UCNP as the control was prepared without Gel-CTX.

Characterization of EcN_IL−2, UCNPs, Gel-CTX, EcN_IL−2@UCNP and EcN_IL−2@UCNP/Gel-CTX

The emission spectra of EcN_IL−2, UCNPs, Gel-CTX, EcN_IL−2@UCNP and EcN_IL−2@UCNP/Gel-CTX were measured by the time-resolved fluorescence spectrometer (FluoroMax+, Japan) with an additional 808 nm laser. Those particles were determined by HT7700 TEM (Hitachi, Japan) at a voltage of 100 kV and the size and zeta potential were tested by Nano Zetasizer ZS90 (Malvern, UK).

Gelatin nanoparticles loaded with FITC (Gel-FITC) and EcN_IL−2 labeled with DiI dye were coupled as described above to obtain EcN_IL−2@UCNP/Gel-FITC. The co-localization of the bacteria and gelatin nanoparticles was observed under a confocal microscope.

The activity of EcN_IL−2@UCNP/Gel-CTX

EcN_IL−2, EcN_IL−2@UCNP, and EcN_IL−2@UCNP/Gel-CTX were incubated at 37°C in LB medium at a volume ratio of 1:100. At five time points (0, 2, 4, 6, and 8 h), 100 µL of diluted culture was spread onto Amp-resistant agar plates, followed by incubation at 37°C for colony imaging and enumeration.

Light-response expression analysis of EcN_IL−2, EcN_IL−2@UCNP and EcN_IL−2-UCNPs in vitro

After irradiation by blue light (475 nm) for 0 min, 30 min, 60 min, 120 min, and 180 min, bacterial suspensions were taken out for detecting the expression of IL-2. The fluorescence intensity of mCherry was then measured using flow cytometry.

EcN_IL−2, EcN_IL−2@UCNP, and EcN_IL−2@UCNP/Gel-CTX bacterial suspensions were prepared for irradiation with NIR light for 30 min, respectively. And then fluorescence images were taken using confocal microscope (Olympus, Japan). One mL of EcN_IL−2 and EcN_IL−2@UCNP bacterial suspensions were added to 1.5 mL Eppendorf tubes, respectively. The EcN_IL−2 group was induced by blue light, while the EcN_IL−2@UCNP group was irradiated with 808 nm NIR at 1 W/cm². The fluorescence signal of mCherry was detected at the different timepoints (0, 30, and 60 min) using IVIS Lumina XR (Caliper Life Sciences, American).

The expression of IL-2 was analyzed by Western blot. Anti-mCherry antibody (Proteintech, China) at the 1:1000 dilution ratio was added into the lysis of blue light-induced EcN_IL−2, NIR-induced EcN_IL−2@UCNP, and their supernatants at 4℃ overnight, respectively. Horseradish peroxidase-conjugated secondary antibody was added and incubated for 1 h at room temperature. Chemiluminescence imaging system (ChemiDoc XRS, Bio-Rad, USA) was used to analyze the electrophoretic bands.

The expression level of IL-2 was further analyzed using a commercial mouse IL-2 ELISA kit. Groups were divided as follows: EcN_IL−2, EcN_IL−2 (NIR+), EcN_IL−2@UCNP, EcN_IL−2@UCNP (NIR+), EcN_IL−2@UCNP/Gel-CTX, and EcN_IL−2@UCNP/Gel-CTX (NIR+), where (NIR+) indicates illumination with NIR light for 2 h. After centrifugation, the bacteria and the supernatant were collected. Subsequently, the content of IL-2 in both the bacteria and the supernatants was detected according to the kit manual, respectively.

EcN_IL−2 and EcN_IL−2@UCNP/Gel-CTX (5 mL, 10⁹ CFU/mL) were respectively added into LB culture medium with Amp (100 µg/mL). After NIR irradiation for 30 min, 150 µL of EcN_IL−2@UCNP/Gel-CTX solution were taken out, and fluorescence signal was detected by flow cytometry at 0 h, 0.5 h, 1 h, and 2 h to investigate the expression level of mCherry-IL-2 fusion protein. The process was repeated for 3 cycles.

Collecting tumor-infiltrating lymphocytes (TILs) and in vitro experiments

One day before tumor engraftment, the right dorsal flanks of 30 male BALB/c mice were clipped and depilated. On day 0, 2 × 10⁶ H22 cells in 100 µL PBS were injected subcutaneously into the same site. When tumors reached 8–10 mm in diameter, they were aseptically excised, diced into 1–2 mm³ fragments, and digested with 0.8 mg/mL collagenase. The digest was passed through a 300-mesh sieve, gently pushed through with a syringe plunger, and washed with PBS to obtain a single-cell suspension.

To isolate tumor-infiltrating lymphocytes (TILs), 5 mL of lymphocyte separation medium was placed in a 15 mL conical tube and overlaid with an equal volume of the tumor single-cell suspension. After centrifugation (3000 rpm, 10 min), four layers were presented from bottom to top: red blood cells, transparent separation medium, diluted buffer, and a whitish lymphocyte layer. The lymphocyte layer was carefully collected, washed three times with washing buffer, and finally resuspended in RPMI-1640 complete medium for use.

Groups were divided as follows: PBS, CTX, EcN_IL−2, EcN_IL−2 (NIR+), EcN_IL−2@UCNP, EcN_IL−2@UCNP (NIR+), EcN_IL−2@UCNP/Gel-CTX (MMP-2+) and EcN_IL−2@UCNP/Gel-CTX (NIR+, MMP-2+). The NIR + group was irradiated with NIR for 30 min each time and then cultured for 2 h, and this process was repeated three times. MMP-2 was added to the MMP-2 + group, and the final concentration was adjusted to 10 µg/mL. At the same time, a blank hole (only adding culture medium) and a control hole (only adding H22 cell suspension) were set, viability using Cell Counting Kit 8 (CCK8) (Dojindo, Japan).

The TILs were co-incubated with bacterial supernatant (filtration with 0.22 μm membrane) after different treatments for 48 h. Cells were collected and stained by differently labeled antibodies. After washing with PBS, the infiltration of CD8⁺ T, Treg and NK cells ratios were analyzed by flow cytometry. CD8⁺ (anti-CD3-FITC, anti-CD8-PE/Cy7), CD4⁺ T cells (anti-CD3-FITC, anti-CD4-Bv421), NK cells (anti-CD49b-APC), and Tregs (anti-CD3-PC/7, anti-CD4-Bv42, anti-CD25-FITC, and then with anti-Foxp3-Alexa Fluor 647 after disrupting nucleus with True-Nuclear™ Transcription Factor Buffer Set) were stained respectively for 30 min. The Gra B within the cells was also measured using a commercial ELISA kit. At the same time, the content of IFN-γ in the supernatant was quantified using a commercial ELISA kit. And then the TILs (1 × 10⁵ cells) treated with different materials were co-incubated with 1 × 10⁴ H22 tumor cells in RPMI 1640 medium without FBS and antibiotics for 5 h. The killing ability of the differently treated TILs against the tumor cells was assessed using an LDH release assay kit.

Animals

Male BALB/c mice (18–20 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Liaoning, China). Mice were housed in cages under constant environmental conditions (22 ± 2°C, 55 ± 5% relative humidity, and a 12 h light-dark cycle). H22 tumor-bearing mice were prepared by subcutaneous injection of H22 cells (2 × 10⁶ cells per mouse) into the flanks of BALB/c mice.

All animals received human care in compliance with the Principles of Laboratory Animal Care Formulated by the National Society of Medical Research. All animal studies were approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology (IACUC Number: 4142).

Biodistribution of EcN_IL−2@UCNP/Gel-CTX

To investigate the distribution of EcN_IL−2@UCNP/Gel-CTX in vivo, IR780-stained EcN_IL−2@UCNP/Gel-CTX (1 × 10⁶ CFU) were injected into H22 tumor-bearing BALB/c mice via i.v. At different time points (0 h, 2 h, 6 h, 12 h, 24 h, 48 h, and 72 h) after injection, fluorescence images were taken by IVIS Lumina XR. The major organs and tumors were collected from mice at 72 h after administration of EcN_IL−2@UCNP/Gel-CTX for fluorescence imaging.

In the meanwhile, mice (three per time point) were intravenously injected via the tail vein with approximately 1 × 10⁶ CFU of EcN_IL−2@UCNP/Gel-CTX. Blood, tumors and other tissues were harvested at 6 h, 12 h, 24 h, 48 h, and 72 h post-injection. After washing with PBS, tissue homogenization was performed using a tissue grinder. The homogenates were temporarily stored at 4℃ and transferred to − 20℃ for long-term preservation. Serial dilutions of the blood and homogenates were prepared, and 100 µL of each dilution was spread onto pre-prepared solid agar plates containing ampicillin using a sterile spreader. The plates were incubated at 37℃ for 24 h, and then the colony-forming units (CFU) on each plate were counted.

Evaluating antitumor effects in vivo

When the volume of tumor achieved to 100–150 mm³, the mice were divided into eight groups: PBS, CTX, EcN_IL−2, EcN_IL−2@UCNP, EcN_IL−2@UCNP/Gel-CTX, EcN_IL−2 (NIR+), EcN_IL−2@UCNP (NIR+), and EcN_IL−2@UCNP/Gel-CTX (NIR+). The mice were injected intravenously with PBS (100 µL), CTX (10 mg/kg in 100 µL), EcN_IL−2 (10⁶ CFU in 100 µL PBS), EcN_IL−2@UCNP (10⁶ CFU in 100 µL PBS), or EcN_IL−2@UCNP/Gel-CTX (10⁶ CFU in 100 µL PBS) 2 times on the 0th and 6th day. NIR light illumination (808 nm, 1 W/cm^− 2, 10 min) was performed on the 2nd, 3rd, 4th, 8th, 9th, and 10th day, respectively. The size of the tumor and the body weight of the mice were measured every day. Tumor volume was calculated by the following formula: 0.5 × length × width².

After illumination on the 14th day, 3 mice per group were sacrificed to obtain lymph node cells for flow cytometry analysis of DC maturation. At the end of treatments, the tumors were weighed and photographed. The major organs (heart, liver, spleen, lung, and kidney) were collected for pathological analysis, and the tumors and spleens were collected for flow cytometry analysis.

Histochemical and Immunofluorescence staining

At the end of experiments, major organs and tumor tissues were collected and fixed in 4% paraformaldehyde for 24 h at room temperature. Pathological changes in heart, liver, spleen, lung, and kidney tissues were detected by H&E staining. Immunohistochemical staining was conducted to determine the expression of Ki67 (a proliferation marker). TUNEL immunofluorescence staining (for detecting apoptotic cells) was carried out in Wuhan Sevicebio Technology Co., Ltd. (Wuhan, China). The images were captured with a light microscope (NIKON, Japan) and a fluorescence microscope, respectively. IFN-γ, Gra B, and Foxp3 in tumor tissues were determined via immunofluorescence assay.

Tumor immune cell analysis in vivo

TDLNs were crushed with a syringe plug to extrude cells and filtered through a 200-mesh nylon mesh, and the cell pellet was collected and resuspended with PBS. Spleens were crushed with a syringe plug and filtered through a 200-mesh nylon mesh, the pellet was resuspended in erythrocyte lysate for 2 min, and then the cell pellet was collected and resuspended with PBS. Tumor tissues were chopped and digested in 1640 medium containing 0.8 mg/mL collagenase at 37 °C for 1 h, then pipetted and filtered through a 300-mesh nylon mesh. The pellet was resuspended with erythrocyte lysate for 2 min, and the cell pellet was collected and resuspended with PBS. In the TDLN group, cells were stained with anti-CD45-APC/cy7, anti-CD3-FITC, anti-CD4-BV421, and anti-CD8-PE/Cy7 for lymphocyte analysis. For DC analysis, cells were stained with anti-CD80-PE, anti-CD86-APC, and anti-CD11c-FITC. In the spleen group, cells were stained with anti-CD3-FITC, anti-CD4-BV421, and anti-CD8-PE/Cy7 for lymphocyte analysis. In the tumor group, cells were stained with anti-CD45-APC/Cy7, anti-CD3-FITC, anti-CD4-BV421, anti-CD8-PE/Cy7, and anti-CD49b-APC for lymphocyte analysis.

Analysis of inflammatory factors in sera and blood biochemistry

At the end of the experiments, blood samples collected from mice were centrifuged (3000 rpm, 10 min) to obtain sera. After dilution with PBS, part of sera was used to measure the levels of TNF-α, IFN-γ, TGF-β and IL-10 by the kits according to the manuals. The other part was used to determine the levels of CREA, AST, ALT, BUN, LDH, and CK using an automatic biochemical analyzer (Mindary, China) in the hospital of Huazhong University of Science and Technology.

Detecting long-term immune memory effects

H22 tumor-bearing mice were divided into eight groups same to the aforementioned antitumor experiment. The mice were administrated intravenously with PBS (100 µL), CTX (10 mg/kg in 100 µL), EcN_IL−2 (10⁶ CFU in 100 µL PBS), EcN_IL−2@UCNP (10⁶ CFU in 100 µL PBS), or EcN_IL−2@UCNP/Gel-CTX (10⁶ CFU in 100 µL PBS) on the 0th, 6th, and 12th day. NIR light illumination (808 nm, 1 W/cm², 10 min) was performed on the 2nd, 3rd, 4th, 8th, 9th, 10th, 14th, 15th and 16th day, respectively. Three mice from each group were sacrificed on the 23rd day, and their spleens were harvested for obtaining a suspension of splenic lymphocytes. Memory T cells in spleens were analyzed by staining with anti-CD3-FITC, anti-CD4-BV421, anti-CD8-PE/Cy7, anti-CD44-PE, and anti-CD62L-PerCP-Cy5.5. To obtain the antigen-specific splenic lymphocytes, the lymphocytes were co-incubated with antigens (80 µg/mL) from H22 cells treated with ultrasound for 72 h. And then antigen-specific splenic lymphocytes were co-incubated with H22 cells for 5 h. The cytotoxic activity of the splenic lymphocytes against the H22 cells was then assessed using a lactate dehydrogenase (LDH) release assay kit.

The tumors were surgically removed from the mice in the EcN_IL−2@UCNP/Gel-CTX (NIR+) group on the 24th day and rechallenged with 2 × 10⁶ H22 cells subcutaneously injected into the opposite backside on the 34th day. The untreated mice at the same age were injected with 2 × 10⁶ H22 cells as controls (n = 5). At the end of experiments (on day 52), mice were sacrificed, and their tumors, spleens, and lymph nodes were harvested, weighed, and photographed.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.0 software, and the results were presented as mean (s. e. m). Two-tailed Student’s T-test or one-way ANOVA followed by Turkey’s test was performed. p < 0.05 was considered to be significantly different. (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, ns: no significant difference.)

Author contributions

Jiawen Chen: Writing – original draft, Methodology, Investigation, Data curation. Renjiang Kong: Writing – original draft, Validation, Methodology, Investigation, Data curation. Yuzhi Qiu: Methodology, Investigation. Yidi Liu: Methodology, Data curation. Xi Yu: Methodology, Data curation. Xiangliang Yang: Project administration, Conceptualization. Yan Zhang: Writing – review & editing, Project administration, Conceptualization. Yanhong Zhu: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Funding

This work was supported by the National Key Research and Development Program of China (2021YFA1101501), and the National Natural Science Foundation of China (No 82172083).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All animal studies were approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology (IACUC Number: 4142).

Consent for publication

All authors of this study agreed to publish.

Competing interests

The authors declare no competing interests.