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. 2024 Apr 17;19(6):481–494. doi: 10.2217/fmb-2023-0210

Akkermansia muciniphila improves gastric cancer treatment by modulating the immune microenvironment

Jianming Fang 1, Huizhong Zhang 1, Xiaodong Zhang 1, Xiaolong Lu 1, Junjie Liu 1, Haiyang Li 1, Jianxin Huang 1,*
PMCID: PMC11216265  PMID: 38629914

Abstract

Background: Gut microbiota is pivotal in tumor occurrence and development, and there is a close relationship between Akkermansia muciniphila (AKK) and cancer immunotherapy. Methods: The effects of AKK and its outer membrane proteins on gastric cancer (GC) were evaluated in vitro and in vivo using cell counting kit-8 assay, flow cytometry, western blotting, ELISA, immunohistochemistry and immunofluorescence. Results: AKK outer membrane protein facilitated apoptosis of GC cells and exerted an immunostimulatory effect (by promoting M1 polarization of macrophages, enhancing expression of cytotoxic T-lymphocyte-related cytokines and suppressing that of Treg-related cytokines). Additionally, AKK and its formulation could inhibit tumor growth of GC and enhance the infiltration of immune cells in tumor tissues. Conclusion: AKK could improve GC treatment by modulating the immune microenvironment.

Keywords: : Akkermansia muciniphila, gastric cancer, immune microenvironment, macrophages

Plain language summary

Akkermansia muciniphila (AKK) is a type of bacteria found in the human gut that is good for the immune system. We wanted to investigate the effect of AKK on cancer. We extracted a protein from AKK called Amuc. AKK and Amuc inhibited the growth of stomach cancer by encouraging the action of immune cells. AKK may therefore be able to treat stomach cancer.

Plain language summary

Summary points.

  • The Akkermansia muciniphila (AKK) outer membrane protein Amuc promoted apoptosis in gastric cancer cells.

  • Amuc increased the number of M1 macrophages and inhibited the number of M2 macrophages.

  • Amuc promoted the expression of macrophage proinflammatory factors IL-23 and TNF-α and inhibited the expression of TGF-β.

  • Amuc promoted macrophage polarization toward M1 and inhibited polarization toward M2.

  • Amuc promoted the expression of cytotoxic T-lymphocyte-associated cytokines IFN-γ and TNF-α, and inhibited the expression of Treg-associated cytokine IL-10.

  • AKK and its preparations significantly inhibited tumor growth.

  • AKK was able to promote CD8+ T immune infiltration and TNF-α secretion.

  • AKK is expected to be an adjuvant regulatory target for gastric cancer immunotherapy.

With over 1 million new cases each year, gastric cancer (GC) is one of the most prevalent malignancies globally and the third biggest cause of cancer-related fatalities [1,2]. Conventional therapies for GC have limited clinical efficacy, and median overall survival for patients with advanced GC is approximately 8 months. Following surgery, chemotherapy, radiation therapy and targeted therapy, immunotherapy has emerged as an optimal clinical option for the treatment of cancer [3]. PD-1 drugs, including nivolumab and pembrolizumab, are now used in the clinical management of advanced GC [4-7]. The results of a phase Ib/II clinical trial (NCT02915432) showed that the use of PD-1 antibody toripalimab in chemotherapy-refractory GC patients resulted in an objective response rate (ORR) of 12.1%, a disease control rate of 39.7%, median progression-free survival of 1.9 months and the median overall survival of 4.8 months [8]. Although advanced GC may benefit from immunotherapy, tumor immune evasion seriously restricts its broad use [9]. It is necessary to further identify new immune regulatory factors and utilize immune modulators to circumvent immune suppression, thereby enhancing the efficacy of GC immunotherapy and optimizing future immunotherapeutic strategies.

Understanding the many traits of complex microbial communities and the probable processes by which microbial communities are engaged in cancer prevention, carcinogenesis and anticancer therapy has become more important over the past 10 years due to the interaction between bacteria and malignancies [10]. Microbial dysbiosis increases cancer susceptibility through multiple pathways. Helicobacter pylori infection, in particular, is linked to an increased risk of gastric adenocarcinoma. Long-term H. pylori infection can lead to atrophic gastritis, intestinal metaplasia, dysplasia and GC, known as the Correa cascade of multistep gastric carcinogenesis [11]. Further research has revealed that the inflammation and immune dysregulation induced by the microbial community and its related metabolites are linked to carcinogenesis. Intestinal bacteria stimulate T-cell responses to bacterial antigens that crossreact with tumor antigens. Specifically, peptide or lipid structures from bacteria can activate a variety of distinct T-cell receptors, thus selecting a surge of T lymphocytes that may be expanded and enter the circulation. These bacterial epitope-specific T cells could recognize crossreactive antigens expressed by normal or cancer cells [12]. In addition to this, intestinal bacteria also cause tumor-specific antigen recognition through small molecule metabolites such as polyamines and short-chain fatty acids that mediate systemic host effects to enhance antitumor immune responses [13]. For instance, in the presence of Bifidobacterium, interferon-related immune genes are increased in secondary lymphoid organ APCs, increasing the effectiveness of anti-PD-L1 therapy [14]. Das et al.[15] demonstrated that H. pylori upregulates PD-L1 in gastric epithelium, and the exposure of gastric epithelial cells to H. pylori suppresses the proliferation of CD4+ T cells from blood, which can be hindered by anti-PD-L1 antibodies. However, the majority of recent investigations on the gut microbiota in GC have concentrated on carcinogenic bacteria, and the possible contribution of helpful bacteria is still largely understood.

The Verrucomicrobia phylum includes the Gram-negative anaerobic bacteria Akkermansia muciniphila (AKK). It can break down mucin and demonstrate powerful adherence to intestinal epithelial cells in the human intestinal mucosa, where it is present [16]. AKK may aid in metabolic health [17] and glucose homeostasis [18]. Additionally, it has been shown to extend the lifespan of premature aging mice [19]. AKK can also alleviate acute and chronic colitis induced by dextran sulfate sodium [20,21]. Furthermore, AKK enhances the effectiveness of cancer immunotherapy via recruitment of CCR9+ CXCR3 + CD4+ T lymphocytes [12] and induction of antigen-specific T-cell responses [22]. These investigations suggest that AKK has the potential to be an effective and biocompatible immune modulator for immunotherapy. However, it remains unclear whether AKK can prevent GC and improve the therapeutic effect on GC tumors via modulation of the tumor immune microenvironment.

We aimed to determine the impact of AKK on the tumor immune microenvironment of GC and its therapeutic effects. We found that AKK outer membrane proteins could promote apoptosis in GC cells and regulate the immune microenvironment via induction of M1 polarization of macrophages and increasing expression of cytotoxic T-lymphocyte (CTL)-related cytokines both in vitro and in vivo, thus suppressing tumor development. These findings supported the protective role of AKK in GC and provided a theoretical basis for using probiotics to increase immunotherapeutic efficacy.

Materials & methods

Cell culture

Human GC cells AGS (BFN607200622) and THP-1 (BFN60700157), as well as PBMC (BFN6072012634), were accessed from Shanghai Cell Bank (China). Human GC cells HGC-27 (CL-0107) were obtained from Procell (Wuhan, China). Cells were cultivated in RPMI 1640 (DearyTech, Shanghai, China) with 10% fatal bovine serum (Vivacell, Shanghai, China), 100 μg/ml streptomycin (DearyTech) and 100 IU/ml penicillin (DearyTech) at 37°C in a 5% CO2 humidified incubator. To induce differentiation into macrophages (Mø), the number of THP-1 cells was adjusted to 1 × 106/ml by adding an appropriate amount of fresh medium. A final concentration of 50 ng/ml of phorbol 12-myristate 13-acetate (Sigma, CA, USA) was added to the cell suspension, gently blown and mixed, and the cell suspension was seeded into a six-well plate at a volume of 2 ml per well and placed in an incubator under light. After 24 h of induction, the cells were harvested and referred to as M⊘[23].

AKK culture & treatment

AKK was obtained from China General Microbiological Culture Collection Center, and Reinforced Clostridial Medium for AKK was purchased from Qingdao Hi-Tech Industrial Park Hope Bio-Technology Co., Ltd (Qingdao, China). Under anaerobic conditions, Columbia blood agar plates (BKMAMLAB, Hunan, China) containing 0.07% (1000 ml) goat blood were used for plate counting to determine the representative CFU/ml. The culture was diluted in phosphate buffer saline (PBS) containing 2.5% glycerol to 1.5 × 108 CFU/100 μl. Subsequently, AKK was heat-inactivated at 70 °C for 30 min [24].

The outer membrane protein of AKK (Amuc) was extracted and quantified using a bacterial outer membrane protein extraction kit (Solarbio, Beijing, China). Specifically, the bacterial suspension was collected, washed twice with PBS, and 500 μl of extraction reagent A was added. The mixture was incubated at 2–8 °C for 1 h with shaking, followed by centrifugation at 12,000× g for 5 min. The supernatant was transferred, heated at 37 °C for 10 min and then centrifuged at 500–1000× g for 3 min. The upper solution was discarded, and approximately 50 μl of the liquid at the bottom of the centrifuge tube was retained. The membrane protein was dissolved in a membrane protein solubilization solution to obtain the AKK outer membrane protein sample. The purified protein sample was quantified using the bicinchoninic acid assay and stored at -80 °C.

Cell counting kit-8 assay

For viability detection, cells (5 × 103/well) were plated into 96-well plates for 24 h. Subsequently, the complete medium was replaced with medium supplemented with varying agents (PBS and Amuc), and cells were further incubated in the incubator for a specific duration (0, 24, 48, 72 h). Then, 10 μl of cell counting kit-8 reagent (GlpBio, CA, USA) was added to each well for 3-h cell incubation. The absorbance was assessed at 450 nm with a microplate reader (Thermo Scientific, MA, USA) [25].

Enzyme-linked immunosorbent assay

Secretion levels of cytokines in the supernatant of different cell cultures were determined by ELISA. Supernatant samples were harvested, and concentrations of IL-23, TNF-α, TGF-β, IFN-γ, TNF-α and IL-10 were quantified with respective standard quantitative ELISA kits (Thermo Scientific). Absorbance values of each sample were measured with a microplate reader (Thermo Scientific) [26].

Western blotting

GC cells were treated with AKK outer membrane protein or PBS. The total protein content was determined using a bicinchoninic acid protein assay kit (Sunny Biotech Hangzhou Co., Ltd, Hangzhou, China) after total proteins were extracted from AGS and HGC-27 cells using RIPA buffer (Solarbio). First, protein samples were separated by SDS-PAGE and transferred to the polyvinylidene fluoride (PVDF) membrane. PVDF membrane was sealed with 10% skim milk (Solarbio) at room temperature for 90 min, rinsed three times with 10 mM Tris-HCl, 150 mM NaCl and 2% Tween-20 (TBST), and incubated at 4 °C with primary antibodies overnight. After removing the PVDF membrane, it was washed three times with TBST for 5 min each time. Subsequently, the membrane was incubated with secondary antibodies at room temperature for 1–2 h and rinsed three times with TBST for 5 min each time. The ECL Ultra-sensitive Chemiluminescence Kit (Beyotime, Shanghai, China) was mixed in a 1:1 ratio to prepare the chemiluminescent substrate. The substrate was evenly applied to the PVDF membrane, and chemiluminescent signals were assayed with a chemiluminescence imaging system, aligning the marker results and protein bands to test the molecular weight of the target protein [27]. Primary antibodies were as follows: rabbit anti-human TRAIL (1:500, ab2056), DR5 (1:1000, ab199357), Cleaved-caspase 8 (1:1000, AF5267), p53 (1:1000, ab32049) and β-actin (1:1000, ab8227); secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000, ab6721). Cleaved-caspase 8 antibody was accessed from Affinity (CO, USA), and other antibodies were accessed from Abcam (Cambridge, UK).

Immunohistochemistry

The tissue samples on slides were deparaffinized and rehydrated with xylene and different concentrations of ethanol. They were then rinsed three times with PBS. Slides were incubated with 3% H2O2 at room temperature for 10 min, followed by sealing with 5% goat serum to hinder nonspecific binding of antibodies. Primary antibodies were applied to slides and incubated at 4 °C overnight. The slides were treated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody for 30 min at 37 °C the next day after being rinsed three times with PBS. Slides were stained with diaminobenzidine and counterstained with hematoxylin. Finally, the slides were dehydrated and mounted. The slides were examined and photographed under an ML31 microscope (MSHOT, Guangzhou, China) [25].

Immunofluorescence

After deparaffinization and rehydration in xylene and gradient alcohols, the sections were rinsed three times in PBS. Sections were blocked with goat serum and incubated with primary antibodies at 4 °C overnight. Following three washes with PBS, sections were incubated with Fluorescein Isothiocyanate (FITC)-labeled anti-mouse antibody (Beyotime) and AF555-labeled anti-rabbit secondary antibody (Beyotime) at 37 °C for 30 min. Subsequently, cell nuclei were counterstained with 4,6-diamino-2-phenyl indole a (Solarbio) for 5 min, followed by mounting with an antifading agent. Sections were photographed and analyzed with a Leica DM2500 LED microscope (Leica, Shanghai, China) [25].

Flow cytometry

For apoptosis analysis, GC cell suspensions were diluted to 105 cells/ml and plated in a six-well plate overnight. AGS and HGC-27 cells were then treated with AKK outer membrane protein or PBS for 24 h. Using the Annexin V-FITC Apoptosis Detection Kit from BestBio Science (Shanghai, China), the proportion of apoptotic cells was calculated. Cells were added to 400 μl of 1× Annexin V Binding Buffer, 5 μl of Annexin V-FITC and 5 μl of propidium. To examine cell apoptosis, a flow cytometer (BD Biosciences, CA, USA) was utilized [28].

For cell cycle analysis, AGS and HGC-27 cells were plated in a six-well plate (105 cells/well). After treatment with AKK outer membrane protein or PBS for 24 h, the cells were rinsed with PBS (8 mM Na2HPO4, 136 mM NaCl, 2 mM KH2PO4, 2.6 mM KCl) and fixed in chilled ethanol for 1 h. After another wash, cells were centrifuged at 1000× g for 5 min. The collected cells were resuspended in 500 μl of PBS containing 20 μl of RNase A from the Cell Cycle Detection Kit (BestBio Science) and incubated at 37 °C for 30 min. Cells were filtered through a cell strainer (pore size: 0.0374 mm) and stained with 400 μl of propidium for 30 min at 4 °C. Flow cytometer was utilized to analyze cells [28].

For macrophage analysis, cells were extracted and stained with fluorescently labeled antibodies, including PerCP anti-human CD68, PE anti-human CD86 (BioLegend, CA, USA), FITC anti-human CD163 (BioLegend) and Fc receptor blocking reagent. The samples were analyzed using a BD FACSVerse flow cytometer (BD Biosciences). Data analysis was conducted using FlowJo_V10 software [29].

CD3+ T-cell sorting

CD3+ T cells were sorted using CD3+ T Lymphocyte Isolation Kit (Thermo Scientific). A total of 50 ml of prechilled cell sorting buffer was added to the PBMCs for washing. The PBMCs were centrifuged at 300× g for 8 min, and the supernatant was removed. An appropriate amount of prechilled cell sorting buffer was added to adjust the PBMC concentration to 1 × 108 cells/ml, and incubated at 4 °C for 5 min. Biotinylated antibodies were added to the PBMCs at a ratio of 20 μl per ml PBMCs. The mixture was gently pipetted up and down 20 times and incubated at 4 °C for 15 min. Next, cells were centrifuged at 300× g for 8 min, and the supernatant was removed. PBMCs were resuspended in cell sorting buffer to a concentration of 1 × 108 cells/ml. Streptavidin-coated magnetic beads were added to the PBMCs at a ratio of 50 μl per ml PBMCs. The mixture was gently pipetted up and down 20 times and incubated at room temperature for 10 min. Cells were mixed with 2.5 ml of cell separation buffer and subjected to magnetic separation for 3 min. The liquid not captured by the magnet was removed. The collected cells were resuspended in 50 ml of prechilled cell sorting buffer and centrifuged at 300× g for 8 min to discard the supernatant. The resulting pellet contained the purified target cells, which were used for downstream analysis. The purified cells were stained with CD3-FITC flow cytometry antibodies and analyzed using a flow cytometer.

Animal experiment

Twelve (n = 3 per group) 4–5-week-old C57BL/6 mice were purchased. After anesthetizing the mice, they were fixed in a supine position on the operating table using medical adhesive tape. The abdominal area of the mice was disinfected with alcohol swabs. The abdominal cavity was opened to expose the stomach, and 100 μl of cell suspension with 1 × 106 murine GC cells MFC (Procell) was injected subserosally into the gastric wall. The successful injection was indicated by the formation of a translucent small bubble under the serosa. The injection site was gently pressed with a cotton swab to prevent leakage, and the stomach was returned to the abdominal cavity. The abdominal cavity was sutured layer by layer, and the incision was disinfected with iodine. After 3 weeks, the mice were orally treated with activated AKK (108 CFU/ml), sterilized AKK, AKK protein Amuc (3 μg) or PBS. During treatment, the condition of the mice was monitored every 3 days. After 3 weeks, mice were euthanized, and gastric tissues were dissected to examine tumor formation. Subsequent immunohistochemistry and immunofluorescence (IF) experiments were performed [24].

Data analysis

All data were presented as mean ± standard deviation and analyzed on Prism software version 7.0. Differences between multiple groups were analyzed by one-way analysis of variance, followed by the least significant difference test for pair-wise comparisons. A p < 0.05 meant statistically significant.

Results

AKK outer membrane protein facilitates apoptosis of GC cells

AKK aids in the maintenance of homeostasis and integrity of the gastrointestinal tract barrier. However, its impact on GC progression has not been studied. We treated GC cells with AKK outer membrane protein (Amuc, 2500 ng/μl) or PBS, and assessed the impact of AKK on the viability of GC cells with cell counting kit-8 assay. Compared with control, AKK outer membrane protein significantly repressed the viability of AGS and HGC-27 cells (Figure 1A). Flow cytometry analysis evaluated the influence of AKK outer membrane protein on apoptosis and the cell cycle of GC cells. Results demonstrated that AKK outer membrane protein significantly promoted apoptosis in GC cells and drove G0/G1 cell cycle arrest in AGS and HGC-27 cells (Figure 1B–C). Western blotting was conducted to measure the expression of apoptosis-associated proteins TRAIL, DR5, Cleaved-caspase 8 and cell cycle regulatory protein p53. Similarly, AKK outer membrane protein was found to enhance protein expression of TRAIL, DR5, Cleaved-caspase 8 and p53 in GC cells (Figure 1D). In conclusion, AKK outer membrane protein fostered apoptosis of GC cells.

Figure 1.

Akkermansia muciniphila outer membrane protein promotes apoptosis in GC cells.

(A) CCK-8 assay for cell viability in GC cells after Amuc treatment. (B) Flow cytometry analysis of cell apoptosis in GC cells after Amuc treatment. (C) Flow cytometry analysis of cell cycle in GC cells after Amuc treatment. (D) WB analysis of the expression levels of apoptosis-related proteins TRAIL, DR5, Cleaved-caspase 8 and cell cycle-regulating protein p53 after Amuc treatment. Data are expressed as the mean ± SD.

*p < 0.05.

AKK: Akkermansia muciniphila; CCK-8: Cell counting Kit-8; GC: Gastric cancer; SD: Standard deviation; WB: Western blotting.

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AKK outer membrane protein promotes infiltration of immune cells in tumor microenvironment

There are reports suggesting that supplementation with AKK can regulate immune cell infiltration in the tumor microenvironment (TME), thus inhibiting the occurrence of colorectal cancer [24]. We treated THP-1 cells with phorbol 12-myristate 13-acetate (0.1 μg/ml) to enhance macrophage differentiation (Mø) after 24 h. The obtained M⊘were then treated with AKK outer membrane protein, and the activation status of macrophages was analyzed using flow cytometry. The results showed that AKK outer membrane protein increased the number of M1 macrophages (CD86 marker) and inhibited the growth of M2 macrophages (CD163 marker) (Figure 2A). Subsequently, ELISA experiments were performed to measure levels of proinflammatory cytokines IL-23 and TNF-α in M1 macrophages, and expression level of TGF-β in M2 macrophages. The results indicated that AKK outer membrane protein significantly promoted the expression of proinflammatory cytokines IL-23 and TNF-α, while inhibiting TGF-β expression, thereby promoting polarization of M⊘toward M1 macrophages and suppressing polarization toward M2 macrophages (Figure 2B–C). CD3+ T cells were isolated from PBMCs (Figure 2D), and AKK outer membrane protein was used to treat activated CD3+ T cells. ELISA tested the expression of CTL-related cytokines IFN-γ and TNF-α, as well as Treg-related cytokine IL-10. The results indicated that AKK outer membrane protein could enhance the expression of CTL-related cytokines and suppress that of Treg-related cytokines (Figure 2E). Therefore, AKK outer membrane protein could promote the infiltration of immune cells in TME.

Figure 2.

Akkermansia muciniphila outer membrane protein promotes immune cell infiltration in the tumor microenvironment.

(A) Flow cytometry analysis of M⊘polarization after Amuc treatment. (B & C) ELISA assay for levels of M1-related cytokines IL-23, TNF-α and M2-related cytokine TGF-β after Amuc treatment. (D) CD3+ T-cell sorting (left: flow cytometry plot before sorting; right: flow cytometry plot after positive sorting). (E) ELISA assay for the expression levels of CTL-related cytokines IFN-γ, TNF-α and Treg-related cytokine IL-10 after Amuc treatment. Data are expressed as the mean ± SD.

*p < 0.05.

AKK: Akkermansia muciniphila; CTL: Cytotoxic T lymphocyte; Mø: Macrophage; SD: Standard deviation; TME: Tumor microenvironment.

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Investigation of therapeutic effects of AKK & its pharmaceutics on GC in mice

To delineate the effects of AKK and its pharmaceutics on GC in vivo, we constructed a mouse in situ xenograft model using murine GC cells (MFC). After 3 weeks, different treatments were administered: AKK live bacteria (AKK-treatment group), AKK sterilized by pasteurization (AKK-treatment group), purified AKK outer membrane protein Amuc (Amuc-treatment group), and PBS-treated tumor-bearing mice as the control group. The results showed that compared with the PBS group, AKK live bacteria significantly repressed tumor growth and AKK sterilized by pasteurization exhibited better therapeutic effects. Moreover, purified AKK outer membrane protein Amuc showed the most optimal effect (Figure 3A). Tumor cell proliferation and growth were highly linked with Ki-67 expression. Subsequently, we used immunohistochemistry to detect the expression of Ki-67, a proliferation marker. AKK live bacteria could significantly suppress Ki-67 expression in tumor tissues. AKK sterilized by pasteurization and purified AKK outer membrane protein Amuc treatment groups showed more pronounced inhibition, with purified AKK outer membrane protein Amuc demonstrating the best inhibitory effect on Ki-67 expression (Figure 3B). To investigate the impact of AKK on immune response, we finally used IF to detect immune cell infiltration level in tumor tissues. It was found that AKK could promote infiltration of CD8+ T cells and stimulate the secretion of TNF-α (Figure 3C–D). Further, IF analysis revealed that AKK could enhance the infiltration ratio of M1 macrophages (labeled with CD80) and inhibit the increase of M2 macrophages (labeled with CD206). Among them, purified AKK outer membrane protein Amuc showed the most significant promotion of CD8+ T cells (secreting TNF-α) and M1 macrophage ratio, as well as the inhibition of M2 macrophage ratio (Figure 3C–E). In conclusion, AKK and its pharmaceutics could restrain the growth of GC tumors in mice and promote infiltration of immune cells in tumor tissues.

Figure 3.

Figure 3.

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Akkermansia muciniphila and its pharmaceutics can inhibit gastric tumor growth in mice and promote immune cell infiltration in the tumor tissue.

(A) Tumor formation in gastric tissues of mice in treatment groups (n = 3). (B) IHC of Ki-67 expression in treatment groups, scale bar = 50 μm. (C) IF of CD8+ T-cell infiltration in tumor tissue of treatment groups, scale bar = 50 μm. (D) ELISA assay for the expression of cytokine TNF-α in treatment groups. (E) IF analysis of macrophage differentiation in treatment groups, scale bar = 50 μm. Data are expressed as the mean ± SD.

*p < 0.05.

AKK: Akkermansia muciniphila; IF: Immunofluorescence; IHC: Immunohistochemistry; SD: Standard deviation.

Discussion

GC has a rising global incidence. In terms of incidence and death, it is one of the top five malignancies globally [3]. In more than 50% of cases, the cancer is discovered after it has spread to other organs. Because advanced GC patients have a dismal prognosis, new methods must be developed to increase the survival rate [30,31]. Among them, targeting immune cells in the GC microenvironment is an effective therapeutic approach. For example, following chemotherapy, modified-Bu-zhong-yi-qi decoction dramatically extends GC patients' survival. The potential anticancer mechanism of modified-Bu-zhong-yi-qi decoction lies in its ability to increase the ratio of CD4+/CD8+ cells, reduce the proportion of CD8+ PD-1+ T cells and PD-1+ Treg cells, and inhibit tumor immune evasion [32]. Lenvatinib is a multikinase inhibitor targeting VEGF receptors and other receptor tyrosine kinases. It can significantly decrease tumor-associated macrophages and increase CD8+ T-cell infiltration, thereby enhancing the antitumor activity of PD-1 inhibitors in in vivo models. Therefore, further exploration of factors that could regulate immune cells in TME of GC is crucial for enhancing immunotherapy against cancer.

Recent studies have shown that with deepening investigations on the immune regulation related to the gut microbiota, it has become possible to use gut microbiota modulation for adjuvant immunotherapy against tumors. By inhibiting immunological checkpoints like PD-1, this strategy hopes to increase action against cancer cells [33,34]. For example, gut microbiota is critical in regulating melanoma patients' response to PD-1 immunotherapy. Patients with a ‘favorable’ gut microbiota composition (highly diverse Ruminococcaceae/Bacillus faecalis) show enhanced antitumor immune responses through improved antigen presentation and enhanced effector T-cell function in TME [35]. AKK treatment facilitated the killing of hepatocellular carcinoma by CXCR6 NK T cells and suppressed the progression of nonalcoholic steatohepatitis to associated hepatocellular carcinoma, according to Li et al. [36]. In non-small-cell lung cancer patients with better immune responses, abundance of AKK is significantly correlated with immune checkpoint inhibitors [37]. AKK strains have been found to improve antitumor activity in germ-free mice transplanted with nonresponsive patient feces and in mice preexposed to antibiotics following treatment with anti-PD-1 drugs [12]. Shi et al. found that combination therapy with IL-2 and AKK effectively recruited a higher proportion of CTLs in tumor-draining lymph nodes, significantly reduced the proportion of Tregs and induced production of proinflammatory cytokines, as demonstrated by significant increases in IFN-γ and TNF-α levels in tumor tissue [38]. Immunosuppressive cytokine TGF-β in serum was reduced, suggesting that IL-2-based immunotherapy with the symbiotic probiotic AKK enhances antitumor immune responses and tumor clearance. This study also found that treatment with AKK outer membrane proteins significantly hindered proliferation, promoted apoptosis in GC cells and enhanced the expression of proinflammatory cytokines IL-23 and TNF-α, which was confirmed in in vivo experiments. Furthermore, gut microbiota can modulate macrophages in TME [39,40]. Macrophages, a vital component of the innate immune system responsible for keeping homeostasis, exhibit high plasticity [41]. M1 macrophages are characterized by their proinflammatory properties and display antitumor activity [42]. They express high levels of inducible nitric oxide synthase and produce a large amount of proinflammatory cytokines, including TNF-α, IL-12 and IL-23 [43]. On the other hand, M2 macrophages primarily participate in anti-inflammatory responses [44]. In TME, macrophages can switch between M1 and M2 phenotypes, and ‘phenotypic switch’ of macrophages is a possible therapeutic target for modulating tumor development [45]. Fan et al. found that AKK suppresses colorectal tumor development by enhancing TLR2/NLRP3-mediated M1 macrophage activation [46]. In prostate cancer, treatment with AKK-derived extracellular vesicles (AKK-EVs) significantly elevated the proportion of CD8+ T cells positive for GZMB+ and IFN-γ+, recruited macrophages, raised the number of M1 macrophages and suppressed the number of M2 macrophages. Conditioned medium from AKK-EV-treated macrophages inhibited proliferation and invasion of prostate cancer cells [25]. Recent research has demonstrated that Amuc_2172 from AKK-EVs reprograms the TME during colorectal carcinogenesis by inducing HSP70 secretion and promoting CTL-associated immune responses, thereby exerting antitumor effects [47]. Similarly, in this study, it was found that AKK outer membrane proteins can promote macrophage polarization toward the M1 phenotype, enhance the expression of CTL-related cytokines IFN-γ and TNF-α, and suppress expression of Treg-related cytokine IL-10.

Recent studies have also found that heat-inactivated AKK may have better metabolic improvement functions compared with pasteurized AKK, suggesting that ‘beneficial’ effects provided by AKK may be mediated through heat-sensitive factors such as proteins [24]. Further research has confirmed that the isolated AKK outer membrane lipoprotein Amuc has the ability to improve metabolism. Amuc may be a key molecule in AKK activation of the TLR2 receptor and can increase production of regulatory T cells and anti-inflammatory cytokines [48]. In other words, both pasteurized AKK and Amuc can partially summarize the beneficial effects of live AKK [49]. Researchers from Nanjing Medical University have made a similar discovery. They have demonstrated that oral administration of pasteurized AKK or its outer membrane protein Amuc can increase the number of CTLs in the colon and mesenteric lymph nodes, upregulate their TNF-α expression, suppress PD-1 expression, and thereby inhibit colitis and colorectal cancer in mice. In coculture experiments of CTL-colorectal cancer cells, pretreatment with Amuc can activate and increase CTLs derived from the spleen, explaining the potential mechanism by which AKK enhances immune efficacy [24]. In addition, Zhu et al. discovered that gavaged Akk may suppress lung tumors by affecting the tumor symbiotic microbiome and reprogramming tumor metabolism [50]. Similarly, in our animal model study, pasteurized AKK and Amuc showed a more significant inhibitory effect on gastric tumor growth in mice, significantly promoted CTL infiltration (expressing TNF-α) and drove infiltration of M1 macrophages while suppressing the number of M2 macrophages, thus promoting immune cell infiltration in the tumor tissue.

This study, however, has some limitations. Firstly, we selected 12 animals for in vivo experiments. Although there are significant differences in the experimental results, the number of experimental animals is small. Both Wang et al. [24] and Luo et al. [25] chose five mice per group for their in vivo studies of AKK. Secondly, clinical trials are needed to confirm the safety and efficacy of AKK or its outer membrane lipoprotein Amuc in mice. Our study provides novel insights and evidence on the regulatory mechanisms of AKK in immune cells in gastric TME, displaying that AKK is promising in clinical disease prevention, diagnosis, monitoring and intervention, and may become an auxiliary regulatory target for GC immunotherapy.

Conclusion

In conclusion, our research results indicated that AKK outer membrane proteins could promote apoptosis in GC cells and had a stimulating effect on tumor immunity. Furthermore, AKK and its pharmaceutics hindered the growth of gastric tumors in mice and drove immune cell infiltration in the tumor tissue.

Funding Statement

This work was supported by the Social Development Project Fund of Jinhua Science and Technology Bureau in Zhejiang Province (2022-3-148).

Financial disclosure

This work was supported by the Social Development Project Fund of Jinhua Science and Technology Bureau in Zhejiang Province (2022-3-148). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

This work was approved by the Experimental Animal Welfare and Ethics Committee of Jinhua Municipal Central Hospital (AL-JHYY202202).

References

Papers of special note have been highlighted as: • of interest; •• of considerable interest

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