微生物学报

Gene name	Forward primers (5′→3′)	Reverse primers (5′→3′)
IL-1β	GCAACTGTTCCTGAACTCAACT	ATCTTTTGGGGTCCGTCAACT
TNF-α	CATCTTCTCAAAATTCGAGTGACAA	TGGGAGTAGACAAGGTACAACCC
IL-6	CCTTCCTACCCCAATTTCCAAT	AACGCACTAGGTTTGCCGAGTA
TGF-β	GAAGGCAGAGTTCAGGGTCTT	GGTTCCTGTCTTTGTGGTGAA
G-CSF	CTCAACTTTCTGCCCAGAGG	AGCTGGCTTAGGCACTGTGT
GM-CSF	GCCATCAAAGAAGCCCTGAA	GTGAAATTGCCCCGTAGACC
HPRT	TTATGGACAGGACTGAAAGAC	GCTTTAATGTAATCCAGCAGGT

Gene name	Forward primers (5′→3′)	Reverse primers (5′→3′)
IL-1β	GCAACTGTTCCTGAACTCAACT	ATCTTTTGGGGTCCGTCAACT
TNF-α	CATCTTCTCAAAATTCGAGTGACAA	TGGGAGTAGACAAGGTACAACCC
IL-6	CCTTCCTACCCCAATTTCCAAT	AACGCACTAGGTTTGCCGAGTA
TGF-β	GAAGGCAGAGTTCAGGGTCTT	GGTTCCTGTCTTTGTGGTGAA
G-CSF	CTCAACTTTCTGCCCAGAGG	AGCTGGCTTAGGCACTGTGT
GM-CSF	GCCATCAAAGAAGCCCTGAA	GTGAAATTGCCCCGTAGACC
HPRT	TTATGGACAGGACTGAAAGAC	GCTTTAATGTAATCCAGCAGGT

地塞米松通过调节中性粒细胞和巨噬细胞向肿瘤的浸润增强减毒鼠伤寒沙门菌介导的肿瘤免疫治疗的抗肿瘤效果

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王珍 ¹ , 朴伶华 ² , 申铉日 ³^,^* , 柳贤德 ¹^,^*

微生物学报 | 研究报告 2024,64(9): 3489-3505

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微生物学报 | 研究报告 2024, 64(9): 3489-3505

地塞米松通过调节中性粒细胞和巨噬细胞向肿瘤的浸润增强减毒鼠伤寒沙门菌介导的肿瘤免疫治疗的抗肿瘤效果

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王珍¹, 朴伶华², 申铉日³^,^*, 柳贤德¹^,^*

作者信息

1 海南大学生命科学学院, 海南海口 570228

2 海南医学院生理学系, 海南海口 571199

3 海南热带海洋学院食品科学与工程学院, 海南三亚 572022

Dexamethasone augments the efficacy of attenuated Salmonella typhimurium-based cancer immunotherapy by modulating the infiltration of neutrophils and macrophages in the tumor microenvironment

Zhen WANG¹, Linghua PIAO², Xuanri SHEN³^,^*, Xiande LIU¹^,^*

Affiliations

1 School of Life Sciences, Hainan University, Haikou 570228, Hainan, China

2 Department of Physiology, Hainan Medical University, Haikou 571199, Hainan, China

3 College of Food Science and Engineering, Hainan Tropical Ocean University, Sanya 572022, Hainan, China

出版时间: 2024-05-29 doi: 10.13343/j.cnki.wsxb.20240157

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摘要

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细菌介导的肿瘤免疫治疗(bacterium-mediated cancer immunotherapy, BCI)在癌症治疗中具有诸多优势, 但地塞米松(dexamethasone, DEX)联合BCI治疗肿瘤的免疫反应机制仍不清晰。

【目的】探究DEX联合减毒鼠伤寒沙门氏菌St.ΔppGpp介导的BCI肿瘤治疗效果及机制。【方法】通过鼠源结直肠癌小鼠模型, 测定St.ΔppGpp+DEX联合治疗的肿瘤抑制效果。使用活体成像测定St.ΔppGpp的肿瘤靶向性与定殖时间。通过伊红(hematoxylin and eosin, H&E)染色, 测试St.ΔppGpp+DEX联合治疗的器官毒性。基于流式细胞术和免疫荧光切片, 测定巨噬细胞极化、中性粒细胞的募集和T细胞反应。通过qRT-PCR, 检测肿瘤微环境中的炎症因子变化。通过人源结直肠癌症小鼠模型, 验证T细胞缺失对St.ΔppGpp+DEX联合治疗的影响。【结果】St.ΔppGpp+DEX联合治疗可显著降低肿瘤大小, 并提高小鼠生存率。DEX可延长St.ΔppGpp在肿瘤细胞的定殖。St.ΔppGpp+DEX联合治疗不会损伤重要免疫器官, 并可促进M2向M1型巨噬细胞极化, 同时抑制中性粒细胞的募集。T细胞缺失不会影响St.ΔppGpp+DEX联合治疗效果。【结论】DEX通过抑制中性粒细胞募集, 增加肿瘤微环境中的M1型巨噬细胞比例, 进而提高减毒鼠伤寒沙门氏菌St.ΔppGpp的抗肿瘤疗效。

关键词

细菌介导的肿瘤免疫治疗 / 鼠伤寒沙门氏菌 / 地塞米松 / 肿瘤微环境 / 细胞免疫反应

Abstract

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Bacterium-mediated cancer immunotherapy (BCI) presents numerous advantages in cancer treatment, while the immune response mechanism of dexamethasone (DEX) combined with BCI for tumor treatment remains unclear. [Objective] To investigate the therapeutic efficacy and mechanism of dexamethasone in combination with attenuated Salmonella typhimurium St.ΔppGpp-mediated BCI. [Methods] The inhibitory effects of St.ΔppGpp+DEX on cancer were evaluated in a murine model of colorectal cancer. In vivo imaging was utilized to determine the tumor targeting and colonization duration of St.ΔppGpp. Organ toxicity resulted from St.ΔppGpp+DEX treatment was assessed by hematoxylin and eosin (H&E) staining. Macrophage polarization, neutrophil recruitment, and T-cell responses were analyzed by flow cytometry and immunofluorescence assay of sections. The changes in inflammatory cytokines in the tumor microenvironment were examined via qRT-PCR. A mouse model transplanted with human colorectal cancer was employed to confirm the effect of T cell depletion on the therapeutic efficacy of St.ΔppGpp+DEX. [Results] The combined treatment St.ΔppGpp+DEX significantly decreased tumor size and enhanced the survival rate of mice. DEX extended the colonization of St.ΔppGpp in tumor cells. Furthermore, St.ΔppGpp+DEX did not induce damage to vital immune organs, and it facilitated the polarization of macrophages from M2 to M1 phenotype while suppressing neutrophil recruitment. T cell depletion did not influence the efficacy of St.ΔppGpp+DEX. [Conclusion] DEX can enhance the anti-tumor effects of St.ΔppGpp by inhibiting neutrophil recruitment and increasing the proportion of M1 macrophages in the tumor microenvironment.

Key words

bacterium-mediated cancer immunotherapy / Salmonella typhimurium / dexamethasone / tumor microenvironment / cellular immune response

引用本文

王珍, 朴伶华, 申铉日, 柳贤德. 地塞米松通过调节中性粒细胞和巨噬细胞向肿瘤的浸润增强减毒鼠伤寒沙门菌介导的肿瘤免疫治疗的抗肿瘤效果. 微生物学报, 2024 , 64 (9) : 3489 -3505 . DOI: 10.13343/j.cnki.wsxb.20240157

Zhen WANG, Linghua PIAO, Xuanri SHEN, Xiande LIU. Dexamethasone augments the efficacy of attenuated Salmonella typhimurium-based cancer immunotherapy by modulating the infiltration of neutrophils and macrophages in the tumor microenvironment[J]. Acta Microbiologica Sinica, 2024 , 64 (9) : 3489 -3505 . DOI: 10.13343/j.cnki.wsxb.20240157

正文

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Cancer immunotherapy is considered a valuable treatment approach for various types of cancer^[1]. The immune checkpoint blockade (ICB) has exhibited remarkable clinical efficacy in patients with melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, or Hodgkin's lymphoma among the various forms of cancer immunotherapy^[2]. ICB works by alleviating the T cell suppression and thereby increasing their tumor cell killing potential^[3]. Despite the overall success of immune checkpoint blockade (ICB), its efficacy has been limited in patients with pancreatic and ovarian malignancies, which are characterized by a low frequency of tumor antigen mutations and a lack of T cell infiltration into the tumor^[4]. Moreover, the interaction between programmed cell death 1 receptor (PD-1) and its ligand PD-L1 has been found to be associated with a range of immune-related adverse effects and cardiotoxicity^[5].

The use of dexamethasone as a first-line prophylactic medication is recommended to effectively mitigate nausea and vomiting in patients undergoing ICB therapy^[6]. In addition, it serves as an immunosuppressant, mitigating the deleterious effects of chemotherapy^[6-8] and ICB treatment regimens^[8-9]. Consequently, the use of dexamethasone in conjunction with other cancer treatments can have a substantial impact on a patient's quality of life and medical expenses. Moreover, dexamethasone has demonstrated potent anti-tumor and anti-angiogenic properties by effectively inhibiting the expression of HIF-1 and vascular endothelial growth factor (VEGF)^[10-11].

The bacteria-mediated cancer immunotherapy (BCI) offers several advantages over ICB, ranging from precise tumor targeting to enhanced tumor penetration^[12-16]. The BCI recruits various immune cells, such as neutrophils, macrophages, and CD4⁺/ CD8⁺ T cells, to the tumor microenvironment^[16-17]. In addition, BCI can directly eradicate tumor cells via bacterial invasion^[18-20]. The genetically modified strain of Salmonella typhimurium (St.∆ppGpp) exhibits a remarkable capacity for proliferation and persistence within tumor tissue, inducing a robust immune response that leads to rapid regression of the tumor^{[14, 21-22]}. The St.∆ppGpp variant has a 10 000 to 1 000 000 times greater 50% lethal dose than the wild-type S. typhimurium strain^[23].

Because Salmonella-mediated cancer therapy cannot completely eradicate tumors alone, it is important to devise an optimal way of combining it with other therapeutic modalities such as chemotherapy or radiotherapy^[24-26]. While Salmonella-mediated cancer therapy and dexamethasone have demonstrated encouraging outcomes independently, the potential anti-tumor efficacy of their combination remains largely unexplored. In addition, whether dexamethasone can modulate the immune response to St.∆ppGpp is uncertain. Therefore, the objective of this study was to investigate the impact of combined treatment using St.∆ppGpp and dexamethasone on both anti-tumor and immune responses in a xenograft mouse model.

1 Materials and Methods

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1.1 Mouse colon cancer model and bacterial injection

To establish the colon cancer xenografts, CT26 cells (1×10⁶/mL; American Type Culture Collection [ATCC]) and HT29 cells (1×10⁶/mL; ATCC) were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (Gibco), and implanted subcutaneously into the right flank of male SPF BALB/c or BALB/c-nu/nu mice (5–6 weeks old; weighing 18–25 g) obtained from Guangdong Medical Laboratory Animal Center (Foshan, China). The tumor growth was monitored on a daily basis by measuring their dimensions with a caliper, and the tumor volume was calculated using the formula (length×height×width)/2. Once the tumor volume reached 120–160 mm³, the mice were randomly divided into four groups (n=9/group): phosphate buffered saline (PBS; vehicle control), dexamethasone (DEX), St.ΔppGpp, or St.ΔppGpp+DEX. The DEX groups received a daily intravenous injection of 0.2 mg/kg dexamethasone, while the St.ΔppGpp groups were administered 3×10⁷ colony forming units (CFU) of St.ΔppGpp bacteria via the tail vein. The mice were euthanized when the tumor volume reached ≥1 500 mm³. All experimental procedures involving mice were approved by the Animal Science and Technology Ethics Committee of Hainan University (No. HNUAUCC-2020-00013) and the mice were anesthetized using either 2% isoflurane or ketamine (200 mg/kg).The specific details are shown in the "Approval Document for Experimental Animal Welfare Ethics Related Projects of Hainan University".

The St.ΔppGpp-Lux, a St.ΔppGpp variant engineered to express the luciferase gene, acquired from Zheng Jinhai at Hunan University. The strain was cultivated in Luria Bertani medium supplemented with kanamycin (Sigma-Aldrich) and preserved as 25% glycerol stocks at a temperature of −80 ℃.

1.2 Optical bioluminescence imaging

When the tumor volume reached 120–180 mm³, the mice underwent intravenous injection of 3×10⁷ CFU of St.ΔppGpp-Lux, followed by bacterial bioluminescence imaging. Subsequently, the mice were randomly divided between two groups: St.ΔppGpp-Lux and St.ΔppGpp-Lux+DEX, with six mice per group. The DEX group received a daily intravenous dose of 0.2 mg/kg dexamethasone. Bioluminescence imaging was performed for 9 consecutive days using an IVIS 100 in vivo imaging system (Caliper).

1.3 H&E staining

The liver, spleen, kidney, and lung tissues were harvested from euthanized tumor-bearing mice and fixed in 4% paraformaldehyde (PFA) solution to evaluate the toxicity of the St.ΔppGpp+DEX combination therapy. Paraffin- embedded tissue sections (3 μm thick) were prepared and subjected to H&E staining using a commercial staining kit (C0105, Beyotime) according to the manufacturer's instructions.

1.4 Quantitative real-time (qRT)-PCR analysis

TRIzol reagent (Beyotime) was used to extract total RNA from tumors. The extracted RNA was reverse transcribed using the SuperScript Ⅱ cDNA Synthesis Kit (TaKaRa) and subjected to qRT-PCR using the SuperReal PreMix Plus Reagent (TIANGEN, China). The primer sequences used in this study are provided in Table 1.

1.5 Flow cytometry

Tumor tissue was immersed in a solution of collagenase Ⅰ (Wuhan Servicebio Technology, Co., Ltd.) and incubated for 45 min at 37 ℃ to extract the cells. Subsequently, the tissue was passed through a 70 µm cell strainer. The cells were then stained with fluorochrome-labeled antibodies targeting CD86, CD206, F4/80, LY6G, CD3, CD4 and CD8 (all from Elabscience Biotechnology, Co., Ltd.) at 4 ℃ for 30 min. A minimum of 10 000 events were recorded on a FACSCalibur flow cytometer (Beckman Coulter CytoFlEX) and the data were analyzed using FlowJo software (Tree Star).

1.6 Zebrafish injury model

The recruitment of neutrophils expressing enhanced green fluorescent protein (eGFP) to the site of injury was visualized in a transgenic (Tg) (mpx: eGFP) zebrafish model (obtained from the China Zebrafish Resource Center, Wuhan, China) using live imaging. To induce the injury, the notochord tip was transected using a sterile scalpel at 72 hour-post-fertilization (hpf), and the number of neutrophils within 0.2 mm of the incision edge was quantified. Damaged tails were treated with dexamethasone (500 μmol/L, Aladdin, China), and the images were captured on an Olympus fluorescence microscope at 4 and 24 hour-post-injury (hpi). The images were analyzed using Image-Pro Plus software.

1.7 Statistical analysis

The statistical analysis was carried out using SPSS 21.0 software. A P-value of less than 0.05 was considered as a measure of statistical significance. The Kaplan-Meier method and log-rank test were used to perform survival analysis. The data were presented as the mean ± standard error of the mean (SEM).

2 Results

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2.1 Dexamethasone enhances the anti- cancer activity of St.∆ppGpp-mediated cancer immunotherapy

Although the efficacy of Salmonella- mediated BCI and dexamethasone was promising, neither monotherapy could fully mitigate tumor development and metastasis in patients^{[19-20, 27]}. Thus, to explore whether dexamethasone could enhance the anti-tumor effect of BCI by regulating the anti-bacterial immune response and remodeling the tumor environment (TME), we examined the potential synergistic effect of the dexamethasone and St.∆ppGpp combination. To this end, mice were intravenously injected with St.∆ppGpp (3×10⁷ CFU) on Day 1, and then treated intraperitoneally with dexamethasone (0.2 mg/kg) from Days 2 to 27 (Figure 1A). The results demonstrated that St.∆ppGpp and dexamethasone exhibited a synergistic anti-tumor effect (versus St.∆ppGpp or dexamethasone alone), which was evidenced by tumor regression (Figure 1B and 1C) and the improved survival rates of model mice (Figure 1D). These results indicate that dexamethasone positively regulates St.∆ppGpp-mediated cancer immunotherapy and that combining St.∆ppGpp with dexamethasone may be a promising anti-cancer strategy.

2.2 Dexamethasone promotes the survival of tumor-colonizing St.∆ppGpp

The success of BCI relies on the ability of therapeutic bacteria to efficiently colonize tumors and induce tumor cell apoptosis and endogenous immune responses^{[16, 28]}. To investigate whether the synergistic antitumor effect of the combination of St.∆ppGpp and dexamethasone is associated with improved survival of tumor-colonized bacteria, this study evaluated the effect of dexamethasone on survival of bioluminescent tumor-colonized St.∆ppGpp-Lux strains. The data of Caliper IVIS Lumina Ⅱ showed that the administration of dexamethasone increased the retention time of St.∆ppGpp-Lux- derived bioluminescence from 10 d (for St.∆ppGpp-Lux alone) to 14 d (Figure 2A). These findings indicate a potential correlation between the capacity of dexamethasone when combined with St.∆ppGpp to augment the antitumor efficacy and the increased survival rates of patients with tumors following treatment involving dexamethasone with St.∆ppGpp.

After observing the retention of St.∆ppGpp in tumors, we assessed the potential impact of combining St.∆ppGpp with dexamethasone on lung, liver, spleen, and kidney tissues in tumor-bearing mice. The data showed that the toxicity of the St.∆ppGpp and dexamethasone combination was similar to that of each therapeutic agent alone. Moreover, the combination therapy did not induce abnormalities, such as steatosis, inflammatory infiltrates, or fibrosis (Figure 2B). This suggests that dexamethasone positively regulates the bacterial colonization of tumors and not that of other organs.

2.3 The effect of dexamethasone on St.∆ppGpp-mediated cellular immune response in the TME

The accumulation of Salmonella in tumor tissue can induce a shift in the tumor microenvironment (TME) from an immunosuppressive state to an immunogenic state by recruiting immune cells, including neutrophils, macrophages, and T cells, into the tumor tissue^{[12, 21, 29]}. The immunogenicity of infected tumors is increased via their apoptosis or through the increased presentation of tumor antigens. This increased tumor antigen release and/or presentation stimulates T cells to kill tumor cells^[30-32]. To assess the impact of dexamethasone on Salmonella-induced anti-tumor cellular immune responses, we employed flow cytometry to quantify the proportions of distinct immune cell populations (specifically neutrophils, M1 and M2 macrophages, as well as CD4⁺/CD8⁺ T cells) within the tumor microenvironment. Hence, the research stained tumor tissues isolated from CT26-tumor-bearing mice with antibodies against F4/80 and CD86 to detect M1-type macrophages; against F4/80 and CD206 to detect M2-type macrophages; against Ly6G to detect neutrophils; against CD3 and CD4 to detect CD4⁺ T cells; and against CD3 and CD8a to detect CD8⁺ T cells. Flow cytometry analysis revealed an increase in the proportion of F4/80⁺CD86⁺ M1-type macrophages (Figure 3A), a decrease in the proportion of F4/80⁺CD206⁺ M2-type macrophages (Figure 3B) and Ly6G⁺ neutrophils (Figure 3C), and no change in the proportions of CD4⁺ T cells (Figure 3D) and CD8⁺ T cells (Figure 3E). The findings suggest that dexamethasone increased Salmonella-mediated anti-cancer activity by decreasing the infiltration of neutrophils and M2-type macrophages and increasing the infiltration of M1-type macrophages into the tumor.

2.4 Dexamethasone decreases St.∆ppGpp- mediated neutrophil infiltration

Being facultative anaerobic bacteria, Salmonella possess a distinctive capability to deeply infiltrate tumor tissue and selectively accumulate in necrotic zones characterized by inadequate vasculature and hypoxia^[33]. However, neutrophils can form a barrier, which restricts the entry of Salmonella into viable cancer cell zones within the tumor. As such, they negatively regulate the ability of these bacteria to effectively colonize tumors, which ultimately reduces the anti-cancer efficacy of BCI^[28]. To investigate the link between dexamethasone, Salmonella retention in tumors, and neutrophil tumor infiltration, immunofluorescence staining was performed on tumor tissue collected from CT26-bearing mice. The results demonstrated that dexamethasone significantly decreased neutrophil infiltration into tumors (Figure 4A and 4B), which was consistent with the findings of the flow cytometry analysis. To further verify the effect of dexamethasone on neutrophils, Tg (mpx: eGFP) zebrafish was used as an inflammatory model^[34-36]. After injuring the zebrafish tail, the recruitment of neutrophils to the injury site was observed by live imaging, and it was found that dexamethasone significantly inhibited the migration of neutrophils to the injury site (Figure 4C). This finding indicates that dexamethasone blocks Salmonella-mediated neutrophil recruitment to the tumor, supporting the flow cytometry and immunofluorescence staining results generated from tumor-bearing mice.

2.5 Dexamethasone increases M1 and decreases M2 macrophage infiltration into St.ΔppGpp-colonized tumors

Tumor-infiltrating macrophages can be categorized into two types: M1, which have anti-tumorigenic activity, and M2, which are pro-tumorigenic. Polarizing M2 macrophages into M1 macrophages represents a promising cancer therapeutic strategy, as it can lead to the transformation of a highly immunosuppressive TME into an immunogenic one^[37-39]. To further characterize Salmonella-mediated macrophage infiltration, immunofluorescence staining was performed on tumor tissues collected from CT26-tumor-bearing mice. The results of the immunofluorescence staining were consistent with those of the flow cytometry analysis, and showed that dexamethasone increased the Salmonella-mediated infiltration of M1 macrophages (Figure 5A and 5B) while decreasing that of M2 macrophages (Figure 5A and 5C) into the tumor, suggesting that the synergistic anti-cancer efficacy of the St.∆ppGpp and dexamethasone combination was associated with increased St.∆ppGpp-mediated M1 macrophage infiltration into the tumor.

2.6 Effect of St.∆ppGpp and dexamethasone on inflammatory cytokines in the TME

Salmonella infection in the TME induces the production of pro-inflammatory cytokines by tumor-infiltrating immune cells, which is associated with Salmonella-mediated anti-tumor efficacy^{[18, 40]}. The expression of various cytokines within the TME was measured by qRT-PCR and it was found that the St.∆ppGpp and dexamethasone combination (versus St.∆ppGpp alone) reduced the expression of GM-CSF, G-CSF, and interleukin (IL)-6 (Figure 5D−5F), while increasing that of IL-1β; the levels of TGF-β and TNF-α were unaltered (Figure 5G−5I). These results suggest a possible link between decreased neutrophil infiltration and the downregulation of genes encoding G-CSF, GM-CSF, and IL-6, as well as between increased M1 macrophage infiltration and the increase in IL-1β expression.

2.7 Dexamethasone enhances the Salmonella- mediated anti-cancer response in a T-cell- independent manner

Because our earlier flow cytometry results showed that dexamethasone did not alter the Salmonella-mediated infiltration of T cells into tumors, we wanted to eliminate the influence of T cells in further experiments. The combination of St.∆ppGpp and dexamethasone was employed in this study to treat HT29-tumor-bearing mice derived from T cell-deficient BALB/c-nu/nu mice (Figure 6A−6C). Similarly, the combined administration of St.∆ppGpp and dexamethasone synergistically enhanced the antitumor efficacy compared to their individual effects. A previous study had indicated that inhibition of the AKT signaling pathway could impede neutrophil recruitment to inflammation sites. To validate the correlation between inhibition of neutrophil recruitment and the Salmonella-mediated anti-tumor effect, we utilized esculetin, an AKT inhibitor, to further suppress neutrophil recruitment to the tumors of T-cell-deficient HT29-tumor-bearing mice. The data showed that the triple combination of St.∆ppGpp, dexamethasone, and esculetin exhibited a significantly enhance anti-tumor effect compared to St.∆ppGpp and either dexamethasone or esculetin alone (Figure 6D and 6E). The findings suggest that targeting the AKT pathway to reduce neutrophil infiltration could be a promising strategy for augmenting the effectiveness of Salmonella-mediated anti-tumor therapy.

3 Discussion

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Salmonella-mediated cancer immunotherapy has certain advantages over traditional tumor immunotherapy, such as effective targeting, tumor penetration, and immune stimulation, coupled with low toxicity and cost^[16]; however, Salmonella alone cannot completely eradicate tumors or inhibit tumor metastasis. Therefore, it is increasingly being used as a combined therapy with radiotherapy and chemotherapy in clinical studies. Combination therapy with conventional drugs, which are already in clinical use and well tolerated by patients, is easier to implement than developing new gene therapies or drugs. In this study, the data showed that dexamethasone significantly enhanced the anti-tumor effect of attenuated Salmonella BCI and significantly prolonged the survival of these bacteria in a tumor-bearing mouse model. Similar results were obtained in another study, which showed that dexamethasone enhanced the anti-tumor efficacy of gemcitabine by promoting the apoptosis and chemosensitivity of tumor cells^[41].

Salmonella exerts three main effects on tumors: (1) it induces apoptosis by infecting tumor cells; (2) its infection of tumor cells leads to the recruitment of more immune cells into the TME; and (3) it increases the production of tumor antigens and the recruitment to antigen-presenting cells such as neutrophils, macrophages, and dendritic cells into the tumor, which ultimately activates T cells to kill tumor cells^{[17, 21, 42]}. Lee et al. reported that the anti-tumor effect of attenuated Salmonella was significantly greater in wild-type mice than in mice lacking CD4⁺ and CD8⁺ T cells, indicating that the therapeutic effect of Salmonella-mediated cancer immunotherapy was directly related to T cell activation^{[21, 30-31]}. The glucocorticoid dexamethasone is frequently employed in clinical practice alongside chemotherapy and radiotherapy to mitigate the adverse effects of these therapeutic modalities through its anti- inflammatory and immunosuppressive mechanisms^[6]; however, dexamethasone itself also exerts an anti-tumor effects by inhibiting tumor angiogenesis^[10-11]. The flow cytometry results generated in the present study showed that dexamethasone had no effect on CD4⁺ and CD8⁺ T cells when used in combination with St.∆ppGpp, and instead increased M1 macrophage and a decreased neutrophil and M2 macrophage infiltration into tumors. The anti-inflammatory role of dexamethasone makes it challenging to provide an explanation for this finding.

Westphal et al. reported that using antibody- mediated systemic inhibition of neutrophil recruitment improved the anti-tumor effect of Salmonella^[28]. The duration of Salmonella retention within tumors has an important impact on tumor apoptosis, immune cell infiltration, and anti-tumor effects. Previous studies have shown that natural products such as chlorogenic acid^[24] and lovastatin^[25] significantly inhibited tumor growth by prolonging the duration of tumor colonization by Salmonella. The data demonstrated that dexamethasone effectively suppressed the recruitment of neutrophils to the injury site in a zebrafish model, which was further validated by flow cytometry and fluorescence immunostaining in tumor-bearing mice treated with St.∆ppGpp. The qRT-PCR results also indicated that G-CSF and GM-CSF production was significantly lower in the tumors of mice treated with St.∆ppGpp and dexamethasone than in those treated with St.∆ppGpp alone; this was associated with the dexamethasone-mediated inhibition of neutrophil recruitment into the tumor. Therefore, it was believed that, by inhibiting neutrophil recruitment, dexamethasone prolongs the retention of St.∆ppGpp within tumors and improves the anti-tumor efficacy of this BCI.

Tumor-associated macrophages (TAM) are divided into the M1 and M2 types: M1 macrophages suppress tumor growth by phagocytosis and cytotoxicity, while M2 macrophages promote tumor cell proliferation and tissue invasion through increased angiogenesis^[43]. These two types of TAMs can be polarized and converted into one another. A study has shown that attenuated St.ΔppGpp carrying flagellin protein B (FlaB) can polarize M2 to M1 macrophages via Toll-like receptor (TLR) 4 and 5 signaling pathways and therefore enhance the anti-tumor activity of Salmonella^[7]. The utilization of flow cytometry and immunofluorescence staining revealed a significant increase in M1 macrophage infiltration and a decrease in M2 macrophage infiltration within the tumors of St.∆ppGpp-treated mice upon administration of dexamethasone. This finding was supported by another study, which showed that dexamethasone influenced the polarization of TAMs from the pro-tumor M2 to the anti-tumor M1 type^[44]. Although the polarization of macrophages was not examined in the present study, we did confirm a direct correlation between the increase in infiltration of M1 macrophages and the synergistic anti-tumor effect observed with the combination of St.∆ppGpp and dexamethasone. The increase in M1 macrophage infiltration can also explain the high expression of pro-inflammatory cytokines (e.g., IL-1β) in the tumors of mice treated with St.∆ppGpp and dexamethasone. Other literature reports also indicated that the IL-1β and TNF-α in tumor infected with therapeutic bacteria were mainly secreted by dendritic cells and macrophages and were closely related to the anti-tumor effect of BCI. Moreover, the restoration of IL-1β and TNF-α levels to pre-treatment levels was associated with tumor recurrence^[40]. Therefore, it was posited that the observed elevation in IL-1β levels in this study can be attributed directly to the infiltration of specific immune cells into the tumor.

To eliminate the influence of T cells on tumor therapy, the utilization of thymectomized nude mice for establishing a tumor model facilitates a more comprehensive investigation into the role played by other types of immune cells. Previous research has demonstrated a close association between AKT phosphorylation and neutrophil migration as well as recruitment^[24-25]. In the HT29 nude mouse model, the combination of dexamethasone and esculetin (an AKT inhibitor) can significantly improved the anti-tumor efficacy of St.∆ppGpp. This result also indirectly demonstrated that the inhibition of neutrophil recruitment to the tumor and the activation of M1 macrophages significantly increased the anti-tumor effect of St.∆ppGpp therapy. Further studies will be needed to explore whether there is a connection between the inhibition of neutrophil recruitment and the activation of M1 macrophages in the TME following St.∆ppGpp treatment.

Overall, the effect of dexamethasone on Salmonella-mediated BCI can be summarized as follows: (1) it exerts anti-tumor effects by inhibiting angiogenesis; (2) it inhibits neutrophil recruitment, thereby promoting tumor colonization by Salmonella; and (3) it directly or indirectly activates M1 macrophages in the TME. Our findings suggest that the combination of dexamethasone and attenuated Salmonella has significant implications for the treatment of cancer patients who lack tumor-infiltrating T cells or are unable to use ICB agents, such as those targeting PD-1 or PD-L1.

基金

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国家自然科学基金(32260854)
海南省科技专项(ZDYF2022SHFZ041)
海南省自然科学基金(821RC529)
海南省自然科学基金(821RC584)
海南热带海洋大学科研基金(RHDRC202314)

参考文献

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参考文献引证文献

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2024年第64卷第9期

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doi: 10.13343/j.cnki.wsxb.20240157

接收时间：2024-03-12
首发时间：2026-03-20
出版时间：2024-05-29

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收稿日期：2024-03-12
录用日期：2024-05-27

基金

National Natural Science Foundation of China(32260854)

国家自然科学基金(32260854)

Science and Technology Special Fund of Hainan Province(ZDYF2022SHFZ041)

海南省科技专项(ZDYF2022SHFZ041)

Natural Science Foundation of Hainan Province(821RC529)

海南省自然科学基金(821RC529)

Natural Science Foundation of Hainan Province(821RC584)

海南省自然科学基金(821RC584)

the Scientific Research Foundation of Hainan Tropical Ocean University(RHDRC202314)

海南热带海洋大学科研基金(RHDRC202314)

作者信息

1 海南大学生命科学学院, 海南海口 570228

2 海南医学院生理学系, 海南海口 571199

3 海南热带海洋学院食品科学与工程学院, 海南三亚 572022

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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