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Parabacteroides distasonis promotes CXCL9 secretion of tumor-associated macrophages and enhances CD8+T cell activity to trigger anti-tumor immunity against anti-PD-1 treatment in non-small cell lung cancer mice

BMC Biotechnology volume 25, Article number: 30 (2025) Cite this article

Abstract

Background

Parabacteroides distasonis (P. distasonis) could regulate inflammatory markers, promote intestinal barrier integrity, and block tumor formation in colon. However, the regulatory effect of P. distasonis on non-small cell lung cancer (NSCLC) remains unknown. This study aimed to investigate the regulatory effect of P. distasonis on NSCLC and its impact on tumor immunity.

Methods

We first established a mouse model of Lewis lung cancer, and administered P. distasonis and intrabitoneal injection of anti-mouse PD-1 monoclonal antibody to assess the impact of P. distasonis on tumor immunity, and mouse intestinal barrier. Then, we explored the effect of P. distasonis on CD8+T cells and CXCL9 secretion mediated by tumor-associated macrophages (TAM). We used the TLR1/2 complex inhibitor CPT22 to evaluate its effect on macrophage activation. Finally, we explored the effect of P. distasonis on CD8+T cells and CXCL9 secreted by TAM in vivo.

Results

In vivo, P. distasonis enhanced anti-tumor effects of anti-PD-1 in NSCLC mice, improved intestinal barrier integrity, recruited macrophages, and promoted M1 polarization. In vitro, CD86 and iNOS levels in BMDM were elevated and CD206 and Arg1 levels were suppressed in membrane fraction of P. distasonis (PdMb) group in comparison to Control group. With additional CPT22 pre-treatment, the levels of CD86 and iNOS in BMDM were reduced, and the levels of CD206 and Arg1 were increased. Compared to PBS group, P. distasonis group exhibited higher proportion of CD8+T cells in tumor tissues, along with increased positive proportion of GZMB and IFN-γ in CD8+T cells. Additionally, in comparison to Control group, PdMb group showed an elevated proportion of GZMB+T and IFN-γ+T cells within CD8+T cells, and secretion of IFN-γ, TNF-α, perforin, and GZMB in CD8+T cell supernatant increased. Moreover, the proportion of CXCL9+F4/80+ macrophages in tumor tissues was higher in P. distasonis group compared to PBS group. In comparison to Control group, CXCL9 protein level in BMDM and CXCL9 secretion level in BMDM supernatant were increased in PdMb group. Finally, P. distasonis enhanced CD8+T cell activity by secreting CXCL9 from macrophages in vivo.

Conclusions

P. distasonis promoted CXCL9 secretion of TAM and enhanced CD8+T cell activity to trigger anti-tumor immunity against anti-PD-1 treatment in NSCLC mice.

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Introduction

Lung cancer ranks as the second most common and deadliest cancer globally [1]. Non-small cell lung cancer (NSCLC) is the most prevalent subtype of lung cancer, characterized by high rate of metastatic spread and drug resistance [2]. Patients with NSCLC currently have access to a range of treatment options, such as surgery, radiotherapy, chemotherapy, molecular targeted therapy, and immunotherapy [3]. However, even with surgical resection, the long-term survival rates for patients with operable NSCLC are still relatively low [4]. Therefore, poor prognosis based on NSCLC requires us to further investigate the molecular mechanisms of NSCLC and develop new and more effective treatments.

Tumor-associated macrophages (TAMs), a prominent type of immune cell found within tumors, play a critical role in establishing an immunosuppressive environment. They achieve this by releasing cytokines, chemokines, growth factors, and immune checkpoint proteins that impede T-cell function [5]. In NSCLC, TAM in the tumor microenvironment (TME) could activate signaling pathways by secreting various cytokines and interacting with other immune cells to promote therapeutic resistance in NSCLC [6]. Research has demonstrated that activated CD8+ T cells have anti-cancer immunity in many different types of cancer [7, 8]. PD-1/PD-L1 axis has been shown to produce inhibitory signals that dampen T cell activity and facilitate tumor immune evasion [9, 10]. However, CXCL9 produced by macrophages is required for anti-tumor immune response after immune checkpoint blockade [11]. Pascual-García et al. revealed that the pleiotropic cytokine LIF could regulate CXCL9 in TAM to prevent tumor infiltration of CD8+T cells into tumors, thereby impacting effectiveness of anti-PD-1 therapy [12]. In addition, Luan et al. reported that blocking C5a receptor could release anti-tumor response of TAM and enhance CXCL9-dependent CD8+T cell activity [13]. Therefore, targeted regulation of TAM phenotypic transformation and CD8+T cell activity may be potential therapeutic strategies for NSCLC.

Recently, probiotics have received a lot of attention for their powerful and well-proven ability to regulate the gut microbiota effectively, thus enhancing gut and overall host health. Exogenous probiotics could generate beneficial metabolites within the body, which can directly or indirectly stimulate and bolster anti-tumor immune response [14]. Researches have shown enrichment or supplementation of Bifidobacterium could enhance the antigen presentation of dendritic cells in TME, further promote infiltration and activation of cytotoxic T lymphocytes within tumors, thereby leading to enhanced effectiveness of immunotherapy that involves PD-1/PD-L1 blockade [15,16,17]. In NSCLC, Akkermansiaceae and Clostridium butyricum have been reported to have therapeutic potential for NSCLC [18, 19]. Parabacteroides distasonis (P. distasonis) serves as a representative strain of the Parabacteroides genus, which comprises Gram-negative anaerobic bacteria frequently found inhabiting the gastrointestinal tract in various species [20]. In previous studies, P. distasonis attenuated TLR4/Akt signaling, regulated inflammatory markers, promoted intestinal barrier integrity, and blocked tumor formation in colon [21, 22]. Additionally, the relative abundance of P. distasonis was associated with long overall survival in NSCLC patients, meaning that P. distasonis was more abundant in NSCLC patients with long overall survival [23]. Furthermore, in NSCLC patients treated with anti-PD-1 blockade therapy, responders have higher levels of P. distasonis [24]. Nevertheless, the regulatory impact of P. distasonis on NSCLC has not been documented, and its effects on TAM and CD8+T cells remain unexplored.

In this study, our aim was to explore the regulatory effect of P. distasonis on NSCLC and unravel the mechanisms underlying its effects on TAM and CD8+T cells through in vivo and in vitro assays. The findings from this study could offer a theoretical foundation for future research on NSCLC immunotherapy and present a novel treatment approach for NSCLC.

Materials and methods

Animals

To explore the effect of P. distasonis on tumor immunity, C57BL/6 male immunocompetent mice (8 weeks of age) were used in this study. Subcutaneously, 2 × 106 Lewis lung cancer cells (LLC, AW-CCM076, derived from the C57BL/6 mouse NSCLC cell line) were inoculated into left axilla of mice to establish a mouse model of Lewis lung cancer [25].

Experiment 1: The mice were randomly divided into four groups: PBS, P. distasonis, αPD-1, and αPD-1 + P. distasonis, five mice in each group. Mice in P. distasonis and αPD-1 + P. distasonis groups were given P. distasonis through oral transplantation for four weeks (2 × 108 CFU/mL dissolved in PBS once daily, 10 mL/kg/day [26]). Mice in the PBS group and αPD-1 group were given the same amount of solvent orally once a day for four weeks. One week after oral administration of P. distasonis, LLC cells were inoculated subcutaneously to establish a Lewis lung cancer mouse model. In each mouse, the number of cells injected was 2 × 106, with an injection volume of 100 µL administered at the left axilla of C57 mice. One week after subcutaneous injection of LLC cells in the left axilla, for ICB treatment, mice from the αPD-1 and αPD-1 + P. distasonis groups were intraperitoneally injected with 200 µg/100 µL anti-mouse PD-1 monoclonal antibody (Clone RMP1-14, RRID: AB_10949053, BioXCell). Mice in PBS and P. distasonis groups were intraperitoneally injected with 200 µg/100 µL IgG (Clone 2A3, RRID: AB_1107769, BioXCell) as isotype control. It was injected once a week starting from day 7 of LLC cell inoculation [27].

Experiment 2: The interaction of CXCL9 and CXCR3 in vivo was interrupted using anti-mouse CXCR3 blocking antibodies. The mice were randomly divided into three groups: αPD-1, αPD-1 + P. distasonis, αPD-1 + P. distasonis + αCXCR3, with five mice in each group. Mice in the αPD-1 + P. distasonis group and αPD-1 + P. distasonis + αCXCR3 group were given P. distasonis via oral transplantation for four weeks (2 × 108 CFU/mL dissolved in PBS, 10 mL/kg/day [26]). Mice in the αPD-1 group were given the same amount of solvent orally once a day for four weeks. For CXCR3 blockade experiment, 200 µg/100 µL anti-mouse CXCR3 monoclonal antibody was injected intraperitoneally into αPD-1 + P. distasonis + αCXCR3 group mice on days 6, 8, 11, 14, 17, and 20 after subcutaneous injection of LLC cells in left axilla [28, 29]. After tumor implantation, tumor length and width were assessed using vernier calipers, and weight was monitored twice a week. The experiment concluded on 21st day when mice were sacrificed. Mice were sacrificed by intraperitoneal injection of sodium pentobarbital (150 mg/kg). Spleen and colon tissues were collected, and subcutaneous tumors were removed and weighed for further analysis.

Culture and preparation of P. distasonis

P. distasonis was cultured in sterile modified peptone-yeast extract glucose medium. All anaerobic microbial work was carried out in anaerobic chambers supplemented with 10% CO2, 10% H2, and 80% N2. The culture was harvested in the logarithmic phase and diluted with PBS to 2 × 108 total colony units/mL [26]. The membrane fraction of P. distasonis (PdMb) was isolated as follows [21]: Cell particles were broken twice on ice by ultrasonic cell crusher, and the membrane components were separated by centrifugation of 8600 g at 4 ℃ for 30 min. P. distasonis and its membrane components were refrigerated at -80 ℃ until use.

Extraction of bone marrow-derived macrophages (BMDMs)

The leg bones of euthanized mice were rinsed with DMEM. Subsequently, cell suspension was filtered via a 70 μm cell filter to eliminate cell aggregates. Red blood cells were lysed via re-suspending precipitate in 2 mL of red blood cell lysate buffer and incubating for 2 min. 1 × 107 bone marrow-derived cells were isolated from femur and tibia of mouse by gradient centrifugation. Bone marrow cells were cultured with 10% FBS in DMEM and treated with 20 ng/mL macrophage colony stimulating factor (m-CSF, 315-02, Peprotech) every other day, and differentiated BMDM was obtained after six days of culture [30].

Isolation and culture of primary CD8+T cells

Mouse spleen was homogenized, and single cells were obtained via centrifugation. Red blood cells were lysed through re-suspending precipitate in 2 mL of red blood cell lysate buffer and incubating for 2 min. Spleen cells were centrifuged, and then suspended at 2 × 106/mL in RPMI-1640 medium with 10% FBS, recombinant IL-2 (10 ng/mL, 212 − 12, Peprotech), β-mercaptoethanol (50 µM, M8210, Solarbio) and anti-CD28 antibody (10312-25, BioGems). Culture dishes were pre-coated with mouse anti-CD3 antibody (05112-25, BioGems). Cells were then cultured for 3–5 days [30]. Spleen T cells were sorted using CD8 T cell isolation kit (130-096-495, Miltenyl Biotec). Cells were prepared and cell numbers were determined. Cell precipitates were re-suspended with 40 µL buffer per 107 cells. Each 107 cells were added with 10 µL Biotin-Antibody Cocktail, thoroughly mixed, and incubated in the refrigerator (2–8 ℃) for 5 min. Each 107 cells were added with 30 µL buffer and mixed well. Each 107 cells were added with 20 µL CD8+T Cell MicroBead Cocktail, thoroughly mixed, and incubated in the refrigerator (2–8 ℃) for 10 min. Magnetic cell separation can be performed after incubation. The column was placed in magnetic field of appropriate MACS separator. Chromatographic column was washed by appropriate buffer, repeatedly rinsing 3 times, and 3 mL each time. Cell suspension was passed through column to collect a eluate containing unlabeled cells, which represented CD8+T cell portion.

Cell culture and treatment

BMDMs were pre-treated with PdMb (100 µg/mL) for 48 h [21], and cultured with IFN-γ (20 ng/mL) and LPS (100 ng/mL) for 24 h to induce polarization into M1 macrophages [30], which were grouped into Control and PdMb. Additionally, BMDMs were pre-treated with PdMb (100 µg/mL) [21] and 10 µM CPT22 for 48 h [31], and cultured with IFN-γ (20 ng/mL) and LPS (100 ng/mL) for 24 h to induce polarization into M1 macrophages [30], which were grouped into Control, Control + CPT22, PdMb, and PdMb + CPT22. Spleen CD8+T cells were cultured for 24 h in BMDM conditioned medium treated or untreated with PdMb (100 µg/mL) induction for 48 h, respectively, and divided into Control and PdMb groups.

Immunohistochemistry (IHC) staining

Ki67, F4/80, iNOS, and CD206 expressions in mouse tumor tissues were assessed via IHC. Sections were toasted, dewaxed to water, and antigens were thermally repaired. Sections were added with 1% periodate for 15 min. Sections were added with Ki67 (AWA10320, Abiowell), F4/80 (70076, CST), iNOS (bs-0162R, Bioss), and CD206 (18704-1-AP, Proteintech) at 4 ℃ overnight. Sections were added with anti-rabbit-IgG antibody-HRP polymers at 37 ℃ for 30 min. Sections were dripped with 50–100 µL preprepared working solution of color developing agent DAB. Sections were restained with hematoxylin for 5–10 min, and returned to blue with PBS. Sections were dehydrated with alcohol. Subsequently, sections were transferred to xylene for 10 min, sealed with neutral gum, and observed under microscope.

Hematoxylin-eosin (HE) staining

HE staining was conducted to evaluate colon tissue injury in mice. Sections were toasted and dewaxed to water. Sections were stained with hematoxylin and returned to blue with PBS. Sections were dyed with eosin and dehydrated with gradient alcohol (or simply baked dry). After removal, sections were transferred to xylene, sealed with neutral gum, and observed under microscope.

Periodic acid-schiff (PAS) staining

Goblet cell number in colon tissue was assessed via PAS staining. Sections were toasted, dewaxed to water, and dripped with 50 µL of periodate to quickly cover the tissue and left for 5–7 min. Sections were stained with Schiff solution, nucleated with hematoxylin for about 20 s, returned blue with PBS, and dried with a hair dryer. Then, sections were dehydrated with gradient alcohol, transferred to xylene, sealed with neutral gum, and observed under microscope.

Western blot

Western blot was utilized to determine ZO-1, Claudin-1, and Occludin levels in mouse colon tissue and CD86, iNOS, Arg-1, and CXCL9 levels in BMDM. RIPA (AWB0136, Abiowell) was utilized to extract total protein. Proteins were separated through SDS-PAGE and transferred to nitrocellulose membrane. Membrane was soaked in 5% skim milk for 1.5 h. Membrane was incubated with ZO-1 (21773-1-AP, 1: 5000, Proteintech), Claudin-1 (AWA41881, 1: 1000, Abiowell), Occludin (27260-1-AP, 1: 3000, Proteintech), CD86 (13395-1-AP, 1: 1000, Proteintech), iNOS (ab178945, 1: 1000, Abcam), Arg-1 (16001-1-AP, 1: 20000, Proteintech), CXCL9 (ab137792, 1: 1000, Abcam), and β-actin (66009-1-Ig, 1: 5000, Proteintech) at 4 ℃ overnight. Secondary antibody was then incubated for 1.5 h. Membrane was incubated with ECL reagent (AWB0005, Abiowell) for 1 min. β-actin was acted as the internal reference protein.

Flow cytometry

First, cells were extracted from tumor tissue. For detection of proportion of tumor-infiltrating macrophages in mouse tumor tissues (CD11b+F4/80+), 1 × 106/100 µL cells were taken into 1.5 mL EP tubes, and F4/80 (12-4801-82, eBioscience) and CD11b (11-0112-82, eBioscience) antibodies were added. For detection of proportion of CD86+ macrophages in tumor tissue (Gated on CD11b+F4/80+), F4/80 (12-4801-82, eBioscience), CD11b (11-0112-82, eBioscience) and CD86 (17-0862-82, eBioscience) antibodies were added. For detection of the positive rate of CD11b+F4/80+CD86+ in BMDM, 1 × 106/100 µL cells were taken into 1.5 mL EP tubes, and F4/80 (17-4801-82, eBioscience), CD11b (11-0112-82, eBioscience) and CD86 (11-0862-82, eBioscience) antibodies were added. Both non-dye tubes and single dye tubes were prepared simultaneously and mixed thoroughly. Cells were then incubated in the dark for 30 min, followed by adding 1 mL PBS for one wash. Cells were centrifuged for 5 min at 350 g and supernatant was abandoned. Cells were washed with 1 mL PBS, centrifuged for 5 min at 350 g, supernatant was abandoned, and finally suspended with 350 µL PBS.

For detection of positive proportion of CXCR3 or GZMB in CD8+T cells in mouse tumor tissue, CD3 (11-0032-82, eBioscience), CD8 (12-0081-82, eBioscience), and CXCR3 (17-1831-82, eBioscience) or GZMB (17-8898-82, eBioscience) antibodies (GZMB need to be fixed and broken film treatment) were added. For detection of proportion of CXCL9+F4/80+ macrophages in mouse tumor tissues, F4/80 (11-4801-82, eBioscience) and CXCL9 (12-3009-80, eBioscience) antibodies (CXCL9 should be fixed and broken) were added. The following steps were the same as the above detection proportion of tumor-infiltrating macrophages in mouse tumor tissues (CD11b+F4/80+). Furthermore, cells were re-suspended with film breaking agent, added into 1.5 mL EP tube, mixed well, and incubated for 30 min. Appropriate amount of buffer was added, and cells were centrifuged at 1000 rpm for 5 min, supernatant was abandoned, and cells were repeatedly washed twice. GZMB or CXCL9 antibody was added, mixed well, and incubated for 30 min. Cells were washed with 1 mL PBS, centrifuged for 5 min at 350 g, supernatant was removed and finally suspended with 350 µL PBS.

For detection of the proportion of GZMB+T cells in primary CD8+T cells of spleen, GZMB (11-8898-82, eBioscience) antibody was added. The following steps were the same as the second half of the steps above for the detection of GZMB positive proportion of CD8+T cells in mouse tumor tissue.

For detection of the positive rate of CD11b+F4/80+CD86+ in BMDM, 1 × 106/100 µL cells were taken into 1.5 mL EP tubes, and F4/80 (17-4801-82, eBioscience) and CD11b (11-0112-82, eBioscience) antibodies were added. Both non-dye tubes and single dye tubes were prepared simultaneously and mixed thoroughly. Cells were then incubated in the dark for 30 min. Cells were centrifuged for 5 min at 350 g, followed by adding 1 mL PBS for one wash. Fixation/Permeabilization concentrate (00-5123-43, eBioscience) was diluted with Fixation/Permeabilization Diluent at a ratio of 1:3 to prepare the 1× working solution. The cell precipitation was suspended by 500 µL 1×Fixation/Permeabilization concentrate, fixed at room temperature for 30 min, centrifuged at 350 g for 5 min, and the supernatant was discarded. 10× Permeabilization Buffer was diluted with deionized water at a ratio of 1:9 to prepare the 1× working solution. 1 mL of 1× Permeabilization Buffer was added to cell precipitation, cells were suspended, centrifuged for 5 min at 350 g, and supernatant was abandoned. The cells were precipitated with 100 µL 1× Permeabilization Buffer, CD206 (53-2061-82, eBioscience) was added and mixed well, and incubated at room temperature for 30 min away from light. The cells were washed once with 1 mL PBS containing 0.5% BSA, centrifuged at 400 g for 5 min at room temperature, then the supernatant was discarded, and the cells were suspended with 150 µL PBS containing 0.5% BSA.

For detection of positive proportion of IFN-γ in CD8+T cells and primary CD8+T cells of spleen, 1 × 106/100 µL cells were taken into 1.5 mL EP tubes, and total volume was adjusted to 500 µL by adding additional medium. Cells were added with 1 µL 500× Cell Stimulation Cocktail (Plus protein transport inhibitors, 00-4975-93, eBioscience) and cultured at 37 ℃ for 4 h. At the end of the treatment, cells were centrifuged for 5 min at 350 g, and supernatant was abandoned. Appropriate amount of CD3 and CD8 antibodies were added, non-dye tube or single dye tube was set, mixed well, and incubated in dark for 30 min. The cells were washed with 1 mL PBS, and centrifuged for 5 min at 350 g. The cell precipitation was suspended by 500 µL 1×Fixation/Permeabilization concentrate and fixed rupture in 30 min in dark. Cells were centrifuged for 5 min at 350 g and supernatant was abandoned. 1 mL of 1× Permeabilization Buffer was added to cell precipitation, cells were suspended, centrifuged for 5 min at 350 g, and supernatant was abandoned and repeated once. Cells were precipitated with 100 µL PBS, added with IFN-γ antibody, mixed well, and incubated for 30 min. Cells were washed with 1 mL PBS, centrifuged for 5 min at 350 g, and supernatant was abandoned.

Quantitative real-time PCR (qRT-PCR)

The colonization of P. distasonis in mouse feces was detected by qRT-PCR. First, the bacterial genomic DNA extraction kit (CW0552S, CWBIO) was used to extract the standard DNA and the standard curve was made. Next, 100–300 mg fecal samples were taken and fecal genomic DNA extraction kit (CW2092S, CWBIO) was applied for fecal DNA extraction. The primer sequences used were: P. distasonis-F: CCACGCAGTAAACGATGA, P. distasonis-R: CTTAACGCTTTCGCTGTG. Additionally, mRNA levels of anti-TAM genes (NOS2, IL-1β, IL-6, IL-12, IL-15 and IL-18) and pro-TAM genes (Arg1, Mrc1, Ym1 and IL-10) were assessed through qRT-PCR. First, Trizol total RNA extraction kit (15596026, Thermo) was utilized to extract total RNA, and the concentration and purity were determined. Then, mRNA was reverse transcribed into cDNA via mRNA reverse transcription kit (CW2569, CWBIO). Gene expression was analyzed on ABI 7900 system with Ultra SYBR Mixture (CW2601, CWBIO). Calculation of gene expression was based on 2−△△Ct method, with β-actin as internal reference gene. Specific primer details can be found in Table 1.

Table 1 The primers used in this study
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Enzyme-linked immunosorbent assay (ELISA)

TNF-α (CSB-E04741m, CUSABIO), IL-6 (CSB-E04639m, CUSABIO), IL-1β (CSB-E08054m, CUSABIO), CXCL9 (CSB-EL006252MO, CUSABIO), IFN-γ (CSB-E04578m, CUSABIO), Perforin (CSB-E13429m, CUSABIO), and granzyme B (GZMB, CSB-E08720m, CUSABIO) kits were conducted to determine TNF-α, IL-6, IL-1β and CXCL9 levels in BMDM supernatant and secretion of IFN-γ, TNF-α, Perforin and GZMB in primary CD8+T cell supernatant of spleen.

Statistical analysis

Statistical analysis was conducted with GraphPad Prism 8.0 software. The measurement data were presented as mean ± standard deviation. Differences between two or more groups were assessed with Student’s t-test or one-way analysis of variance (ANOVA). Statistical significance was considered at P < 0.05.

Results

P. distasonis enhanced anti-tumor effect of anti-PD-1 in NSCLC mice

To explore the impact of P. distasonis on tumor immunity, we first established a mouse model of Lewis lung cancer and administered P. distasonis by oral transplantation and intraperitoneal injection of 200 µg/100 µL anti-mouse PD-1 monoclonal antibody. First, we detected the colonization of P. distasonis in mouse feces by qRT-PCR. The results showed that, compared with the PBS group, the level of P. distasonis was significantly increased in the P. distasonis group (Fig. 1A). Figure 1B showed images of mouse tumors. We observed that there were no significant alterations in tumor volume and weight between P. distasonis group and PBS group, whereas tumor volume and weight decreased in αPD-1 group. However, compared with P. distasonis group or αPD-1 group, tumor volume and weight of mice in αPD-1 + P. distasonis group were further reduced (Fig. 1C and D). In addition, compared with PBS group, Ki67 expression in tumor tissues of P. distasonis group was not significantly changed, and Ki67 expression in αPD-1 group was decreased. However, compared with P. distasonis group or αPD-1 group, Ki67 expression in αPD-1 + P. distasonis group was further decreased (Fig. 1E). These results suggested that P. distasonis enhanced anti-tumor effect of anti-PD-1 in NSCLC mice.

Fig. 1
figure 1

P. distasonis enhanced anti-tumor effect of anti-PD-1 in NSCLC mice. (A) Detection of P. distasonis colonization in mouse feces by qRT-PCR. (B) Tumor images. (C) Tumor volume growth curve. (D) Tumor weight. (E) IHC staining of Ki67 expression in mouse tumor tissue. Scale bar = 100 μm (100×), Scale bar = 25 μm (400×). *P < 0.05 vs. PBS, &P < 0.05 vs. P. distasonis, #P < 0.05 vs. αPD-1

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P. distasonis improved intestinal barrier integrity in NSCLC mice

To explore the impact of P. distasonis on intestinal barrier of NSCLC mice, we detected the colon tissue injury through HE staining. Colonic epithelial cell morphology and structure in PBS group were intact, but there were some apical villous submucosal spaces. After P. distasonis treatment, colon mucosal epithelial cell morphology and structure were complete and orderly, with no obvious cell proliferation, and the structures of muscle layer and serosal layer were intact (Figure S1A). In addition, PAS staining revealed a higher number of goblet cells in colon tissue of P. distasonis group compared to PBS group (Figure S1B). The detection of tight junction protein showed that compared with PBS group, ZO-1, Claudin-1 and Occludin levels in colon tissue of mice in P. distasonis group were increased (Figure S1C and S2). These results indicated that P. distasonis improved intestinal barrier integrity in NSCLC mice.

P. distasonis recruited macrophages and promoted M1 polarization

To explore the impact of P. distasonis on tumor-infiltrating macrophages, we first detected the proportion of tumor-infiltrating macrophages (CD11b+F4/80+) in mouse tumor tissues by flow cytometry. We observed a markedly elevate in proportion of tumor-infiltrating macrophages (CD11b+F4/80+) in the P. distasonis group compared to PBS group (Fig. 2A). To demonstrate the effect of P. distasonis on the M1/M2 macrophages in vivo, we presented a flow cytometry gating strategy to identify total macrophages and M1 macrophages through detecting infiltrating macrophages in tumor tissue of NSCLC mice (Figure S3A). Additionally, mRNA levels of anti-TAM genes (NOS2, IL-1β, IL-6, IL-12, IL-15, and IL-18) were elevated in P. distasonis group compared to PBS group, while mRNA levels of pro-TAM genes (Arg1, Mrc1, Ym1, and IL-10) were reduced (Fig. 2B and C). Moreover, compared with PBS group, the proportion of CD86+ macrophages in P. distasonis group, F4/80 and iNOS expressions in tumor tissues increased, but CD206 expression decreased (Fig. 2D and E). Our results suggested that P. distasonis recruited macrophages and promoted M1 polarization.

Fig. 2
figure 2

P. distasonis recruited macrophages and promoted M1 polarization. (A) Flow cytometry detection of proportion of tumor infiltrating macrophages (CD11b+F4/80+) in mouse tumor tissues. (B) mRNA levels of anti-TAM genes (NOS2, IL-1β, IL-6, IL-12, IL-15, and IL-18) in mouse tumor tissues. (C) mRNA levels of pro-TAM genes (Arg1, Mrc1, Ym1, and IL-10). (D) Flow cytometry detection of the proportion of CD86+ macrophages in tumor tissue (Gated on CD11b+F4/80+). (E) IHC staining of F4/80, iNOS and CD206 expressions in mouse tumor tissues. Scale bar = 100 μm (100×), Scale bar = 25 μm (400×). *P < 0.05 vs. PBS

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P. distasonis promoted the M1 polarization in vitro

Next, we further explored the impact of P. distasonis on macrophages at cellular level. BMDM was extracted from Lewis lung cancer mice, pre-treated with 100 µg/mL PdMb for 48 h, and cultured with 20 ng/mL IFN-γ and 100 ng/mL LPS for 24 h to induce polarization into M1 macrophages. In comparison to Control group, CD11b+F4/80+CD86+ positive rate, CD86 and iNOS levels in BMDM in PdMb group were increased, while CD11b+F4/80+CD206+ positive rate and Arg1 levels were decreased (Fig. 3A and B, Figure S2). Figure S3B and S3C showed the images of flow cytometry gating strategy to identify M1 macrophages and M2 macrophages through detecting the polarization phenotype of BMDM. In addition, compared to Control group, TNF-α, IL-6, and IL-1β levels in BMDM supernatant in PdMb group were increased (Fig. 3C). Our results demonstrated that P. distasonis promoted M1 polarization of macrophages in vitro.

Fig. 3
figure 3

P. distasonis promoted the M1 polarization of macrophages in vitro. (A) Flow cytometry analysis of the positive rate of CD11b+F4/80+CD86+ and CD11b+F4/80+CD206+ in BMDM. (B) Western blot analysis of CD86, iNOS, and Arg1 expressions in BMDM. (C) ELISA assessment of TNF-α, IL-6 and IL-1β levels in BMDM supernatant. *P < 0.05 vs. Control

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TLR1/2 was involved in the recognition of P. distasonis by macrophages in vitro

It has been reported that TLR1 and TLR2, as heterodimers, are involved in the recognition of microorganisms [32]. Therefore, we used the TLR1/2 complex inhibitor CPT22 [33] to assess its impact on macrophage activation. BMDM was extracted from Lewis lung cancer mice, pre-treated with 100 µg/mL PdMb [21] and 10 µM CPT22 [31] for 48 h, and cultured with 20 ng/mL IFN-γ and 100 ng/mL LPS for 24 h to induce polarization into M1 macrophages. Compared to the Control group, the positive rate of CD11b+F4/80+CD86+ and the levels of CD86 and iNOS in BMDM of the PdMb group were increased, while the positive rate of CD11b+F4/80+CD206+ and the levels of Arg1 were decreased. With additional CPT22 pre-treatment, the positive rate of CD11b+F4/80+CD86+ and the levels of CD86 and iNOS in BMDM were reduced, but the positive rate of CD11b+F4/80+CD206+ and the levels of Arg1 were increased (Fig. 4A and B, Figure S2). Furthermore, compared to the Control group, the levels of TNF-α, IL-6, and IL-1β in the supernatant of BMDM in the PdMb group were increased. With additional CPT22 pre-treatment, the levels of TNF-α, IL-6, and IL-1β in the supernatant of BMDM were decreased (Fig. 4C). Our results indicated that TLR1/2 was involved in the recognition of P. distasonis by macrophages in vitro. Blocking of TLR1/2 partially inhibited the recognition of P. distasonis by macrophages, suggesting the presence of other immune recognition mechanisms.

Fig. 4
figure 4

TLR1/2 was involved in the recognition of P. distasonis by macrophages in vitro. (A) Flow cytometry analysis of the positive rate of CD11b+F4/80+CD86+ and CD11b+F4/80+CD206+ in BMDM. (B) Western blot analysis of CD86, iNOS, and Arg1 expressions in BMDM. (C) ELISA assessment of TNF-α, IL-6 and IL-1β levels in BMDM supernatant. *P < 0.05 vs. Control, & P < 0.05 vs. PdMb

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P. distasonis enhanced TAM-mediated CD8+T cell activity

Next, we explored the impact of P. distasonis on CD8+T cells at animal level and cellular level. Compared with PBS group, the positive proportion of GZMB and IFN-γ in CD8+T cells elevated (Fig. 5A and B). Additionally, splenic CD8+T cells were cultured for 24 h in BMDM-conditioned medium that was either treated or untreated with 100 µg/mL PdMb for 48 h. Compared to Control group, PdMb group exhibited an elevated proportion of GZMB+T and IFN-γ+T cells within CD8+T cells. Moreover, there was an elevation in secretion of IFN-γ, TNF-α, perforin, and GZMB in CD8+T cell supernatant (Fig. 5C and E). Our results suggested that P. distasonis enhanced TAM-mediated CD8+T cell activity.

Fig. 5
figure 5

P. distasonis enhanced TAM-mediated CD8+T cell activity. (A and B) Flow cytometry analysis of positive proportion of GZMB and IFN-γ in CD8+T cells in mouse tumor tissue. *P < 0.05 vs. PBS. (C and D) Flow cytometry determination of proportion of GZMB+T and IFN-γ+T cells in primary CD8+T cells of spleen. (E) ELISA detection of secretion of IFN-γ, TNF-α, Perforin and GZMB in supernatant of primary CD8+T cells of spleen. *P < 0.05 vs. Control

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P. distasonis enhanced TAM-mediated CXCL9 secretion

Research has shown that blocking C5a receptor could release anti-tumor response of TAM and enhance CXCL9-dependent CD8+T cell activity [13]. Here, we also wanted to examine the effect of P. distasonis on TAM-mediated CXCL9 secretion. We observed the proportion of CXCL9+F4/80+ macrophages in tumor tissues increased in P. distasonis group compared to PBS group (Fig. 6A). In addition, in comparison to Control group, CXCL9 protein levels in BMDM and CXCL9 secretion levels in BMDM supernatant were increased in PdMb group (Fig. 6B and C, Figure S2). Our results indicated that P. distasonis enhanced TAM-mediated CXCL9 secretion.

Fig. 6
figure 6

P. distasonis enhanced TAM-mediated CXCL9 secretion. (A) Flow cytometry detection of the proportion of CXCL9+F4/80+ macrophages in mouse tumor tissues. *P < 0.05 vs. PBS. (B) Western blot assessment of CXCL9 protein levels in BMDM. (C) ELISA analysis of the secretion level of CXCL9 in BMDM supernatant. *P < 0.05 vs. Control

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P. distasonis enhanced CD8+T cell activity by secreting CXCL9 from macrophages in vivo

Finally, we explored the impact of P. distasonis on CD8+T cells and CXCL9 secreted by TAM in vivo. As shown in Fig. 7A, compared to the PBS group, the proportion of CXCR3 in CD8+T cells in the tumor tissue of the P. distasonis group increased, suggesting that promoting the recruitment of CD8+T cells to the tumor through CXCR3 enhanced antitumor immunity. Figure 7B displayed photos of tumor in mice. Tumor volume and weight of αPD-1 + P. distasonis group were decreased compared with αPD-1 group. The interaction of CXCL9 and CXCR3 in vivo was interrupted using anti-mouse CXCR3 blocking antibodies. Using αCXCR3 resulted in increased tumor volume (Fig. 7C and D). In addition, compared with αPD-1 group, αPD-1 + P. distasonis group exhibited decreased Ki67 expression in mouse tumor tissues but elevated positive proportion of GZMB and IFN-γ in CD8+T cells. After use of αCXCR3, Ki67 expression in mouse tumor tissues increased, while positive proportion of GZMB and IFN-γ in CD8+T cells also decreased (Fig. 7E and G). Our results showed that P. distasonis enhanced CD8+T cell activity by secreting CXCL9 from macrophages in vivo.

Fig. 7
figure 7

P. distasonis enhanced CD8+T cell activity by secreting CXCL9 from macrophages in vivo. (A) Flow cytometry determination of positive proportion of CXCR3 in CD8+T cells in mouse tumor tissue. *P < 0.05 vs. PBS. (B) Tumor images. (C) Tumor volume growth curve. (D) Tumor weight. (E) IHC staining of Ki67 expression in mouse tumor tissue. Scale bar = 100 μm (100×), Scale bar = 25 μm (400×). (F and G) Flow cytometry determination of positive proportion of GZMB and IFN-γ in CD8+T cells in mouse tumor tissue. *P < 0.05 vs. αPD-1, &P < 0.05 vs. αPD-1 + P. distasonis

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Fig. 8
figure 8

P. distasonis promoted the secretion of CXCL9 by TAM and enhanced activity of CD8+T cells, triggering anti-tumor immunity against anti-PD-1 therapy in NSCLC mice

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Discussion

In recent years, immunotherapy has revolutionized cancer treatment, but most patients are difficult to cure or develop resistance to immunotherapy [34]. Therefore, a deeper understanding of the mechanisms of NSCLC and tumor immunity may help in the search for new treatments. In this research, we investigated the mechanism of P. distasonis in NSCLC primarily at the animal and cellular levels, where it was involved in tumor immunity. We found that P. distasonis promoted the secretion of CXCL9 by TAM and then enhanced activity of CD8+T cells, triggering anti-tumor immunity against anti-PD-1 therapy in NSCLC mice (Fig. 8). This is also the first time we report on P. distasonis in NSCLC.

Recently, the gut microbiome has sparked a great deal of enthusiasm for cancer immunotherapy. Under physiological conditions, intestinal mucosal barrier, gut microbiota and their metabolites jointly form intestinal microecology and maintain dynamic balance [35]. Dysfunctional gut microbiota is associated with inefficient cancer treatments [36, 37], while a “healthy” gut microbiota is often associated with positive outcomes from immunotherapy [38]. Immunotherapy represents a groundbreaking approach in the realm of cancer treatment, aimed at boosting host immune system to elicit anti-tumor effects [39]. Among the various immunotherapeutic strategies, immune checkpoint blockade has shown significant advancements in treating a range of malignant tumors [40]. Immune checkpoint blocking therapy could block negative immunomodulators in TME. This approach helps enhance tumor immune surveillance and boosts the host immune response against tumors [41, 42]. Therefore, the regulation of the gut microbiota in conjunction with immune checkpoint inhibition holds promise as a potent strategy for advancing a new era of anti-tumor therapies. Probiotics and microbial-mediated therapeutics also represent potential avenues to augment traditional anti-tumor treatments [43]. P. distasonis plays a critical role in human health, impacting conditions including diabetes, colorectal cancer, and inflammatory bowel disease [44]. In A/J mice treated with azoxymethane, P. distasonis slowed tumorigenesis, regulated inflammatory markers, and promoted intestinal barrier integrity [22]. Furthermore, P. distasonis combined with α-PD-1 monoclonal antibody demonstrated a markedly delay in bladder cancer growth and an elevate in density of CD4+T and CD8+T cells within tumor. In other words, combination administration of P. distasonis with α-PD-1 monoclonal antibody could represent a novel strategy to strengthen effectiveness of anti-PD-1 immunotherapy by activating immune and anti-tumor pathways [45]. In a clinical study involving lung cancer patients, it was observed that the responders to PD-1 blockade exhibited higher abundance levels of P. distasonis and Bacteroides vulgatus compared to non-responders [24]. In this study, we employed a subcutaneous xenograft model of NSCLC, in which the tumor tissue was located subcutaneously and was physically isolated from the gut. According to existing literature, orally administered probiotics typically colonize the gut, reshape the gut microbiota, and subsequently trigger anti-tumor responses [46,47,48]. We found that the colonization of P. distasonis in mouse feces in the P. distasonis group was increased compared with the PBS group. Therefore, we speculate that P. distasonis may indirectly modulate the tumor microenvironment via the “gut-tumor” axis, rather than directly colonizing tumor tissues. We revealed that P. distasonis enhanced anti-tumor effect of anti-PD-1 in NSCLC mice and improved intestinal barrier integrity in NSCLC mice. Therefore, we would further explore the mechanism of P. distasonis in tumor immunity.

TME is a complex system in which malignant cells interact individually or in combination with immune and non-immune cells to affect sensitivity to immunotherapy [49]. Macrophages can be divided into two extreme subgroups, classically activated (M1) and alternately activated (M2) macrophages, which exhibit distinct functional characteristics in response to microenvironmental stimuli. In TME, TAM is considered to be a polarized M2 phenotype that promotes tumor progression and has a poor prognosis [50]. IFN-γ, L-1β, and LPS induce macrophages (M1), whereas IL-4 and IL-13 trigger macrophages (M2) [51]. The polarization and infiltration of TAM play an essential role in initiation and progression of malignant tumors like lung cancer and tumor immune microenvironment [52]. In our research, we found that P. distasonis recruited macrophages and promoted M1 polarization in vivo. In vitro, we further demonstrated that P. distasonis promoted M1 polarization of macrophages. Moreover, TLR1/2 was involved in the recognition of P. distasonis by macrophages in vitro. Blocking of TLR1/2 partially inhibited the recognition of P. distasonis by macrophages, suggesting the presence of other immune recognition mechanisms. Therefore, gaining a more profound understanding of the intricate role of TAM in immunotherapy regulation could provide new insights into TME. Next, we wanted to delve deeper into the role of P. distasonis in CD8+T cells.

The presence of activated CD8+T cells in the peritumoral stroma has been demonstrated to hold significant positive prognostic value [53]. More and more clinical evidence showed that tumor-infiltrating CD8+ lymphocytes within tumor environment were correlated with the survival outcomes of cancer patients. This further underscores the intimate relationship between immune evasion and TME [54, 55]. Dysfunction of anti-tumor CD8+T cells occurs when inhibitory receptor PD-1 is activated by PD-L1 expressed on tumor cells (or other cells in the TME). Blocking interaction between PD-1 and PD-L1 with specific monoclonal antibodies may restore the function of CD8+T cells, leading to tumor regression [56]. Jing et al. reported that integrin α2 repressed CD8+T cell activity through upregulating exosomal PD-L1 expression and promoted immune escape of NSCLC [57]. In NSCLC, the impact of P. distasonis on CD8+T cells remains unknown. In this research, it was discovered that P. distasonis enhanced TAM-mediated CD8 + T cell activity at both animal and cellular levels. Our study revealed a novel mechanism between P. distasonis and TAM-mediated CD8+T cells in NSCLC.

CXCL9 can be used to predict postoperative recurrence in patients with NSCLC after surgery [58]. CXCL9/10-engineered dendritic cells promoted T cell activation and enhanced lung cancer immune checkpoint blocking [59]. In addition, irradiation mediated IFN-α and CXCL9 expressions in NSCLC, stimulating CD8+T cell activity and tumor migration. Among them, IFN-α mainly elevated IFN-γ levels in CD8+T cells and collaborated with CXCL9 to promote CD8+T cell migration in vitro [60]. Therefore, in the research, we also wanted to investigate the impact of P. distasonis on TAM-mediated CXCL9. We found that P. distasonis enhanced TAM-mediated CXCL9 secretion at both animal and cellular levels. Finally, we used anti-mouse CXCR3-blocking antibodies to disrupt the interaction between CXCL9 and CXCR3 in vivo. We found that P. distasonis enhanced CD8+T cell activity by secreting CXCL9 from macrophages in vivo. The expression of CXCR3 is variable across different types of tumor cells. For example, in glioblastoma, CXCR3-A is highly expressed, contributing to tumor proliferation and invasion, while CXCR3-B is expressed at lower levels with limited functional impact [61]. Similarly, in other cancers such as renal cell carcinoma and breast cancer, CXCR3-B is associated with apoptosis and vascular stasis [62, 63]. CXCR3 is widely expressed in immune cells, including T cells, dendritic cells, and natural killer cells [64]. In activated T cells, CXCR3-A is the predominant subtype, facilitating the recruitment of CD4+ and CD8+ T cells to the tumor site [65]. The expression of CXCR3 on regulatory T cells within the tumor has also been shown to suppress CD8+ T cell anti-tumor immunity [66]. CXCR3 ligands (CXCL9, CXCL10, and CXCL11) are primarily secreted by endothelial cells, fibroblasts, and certain immune cells, recruiting CXCR3-positive immune cells into the tumor microenvironment [67, 68]. Moreover, TAMs play a crucial role in shaping the tumor microenvironment and can influence the expression and function of CXCR3-positive cells [69, 70]. For instance, TAMs can secrete CXCL9, regulating the recruitment of CXCR3-expressing CD8+ T cells, thereby affecting the response to anti-PD-L1 therapy [71]. Additionally, TAMs may indirectly modulate the CXCR3 pathway by altering the expression of CXCR3 ligands or affecting the activation state of CXCR3-positive immune cells [72]. In summary, we have incorporated these findings to highlight the distinct expression patterns of CXCR3 across different cell types and the potential regulatory role of TAMs in modulating CXCR3-positive cells within the tumor microenvironment. Our study confirmed that the novel mechanism of P. distasonis in NSCLC is related to TAM-related immunity.

However, we are currently unable to conduct metagenomic studies on tumor samples to detect the presence of P. distasonis or perform any specific staining for P. distasonis. In the future, we plan to use an in situ lung cancer model (such as intrapulmonary injection) combined with metagenomic sequencing for further investigation. Furthermore, we plan to include an analysis of CXCR3-positive cell subpopulations in our subsequent studies. We will use flow cytometry to sort CXCR3-positive cells from tumor tissues and combine this with multiplex immunofluorescence staining techniques to analyze their co-expression with other cell markers (such as CD3, CD8, CD4, CD11c, etc.), in order to clarify the distribution of different cell subpopulations. Additionally, through single-cell RNA sequencing, we will conduct a more in-depth classification of CXCR3-positive cell subpopulations to reveal their heterogeneity and potential functions within the tumor microenvironment. Furthermore, by combining immunohistochemistry and immunofluorescence techniques, we will perform co-staining of CXCR3 and related subpopulation markers on tumor tissue sections to visually demonstrate the spatial distribution of CXCR3-positive cell subpopulations within the tumor tissue.

Together, our findings suggested that P. distasonis might ameliorate NSCLC. Through in vivo and in vitro studies, we confirmed that P. distasonis promoted the secretion of CXCL9 by TAM, thereby boosting function of CD8+T cells and activating anti-tumor immunity in response to anti-PD-1 therapy in NSCLC mice. In the future, further clinical trials and experimental studies are needed to enhance our understanding of the regulatory mechanisms of P. distasonis. Our study serves as a valuable reference and foundation for future clinical interventions in NSCLC, aiding in the expansion of novel treatment approaches for this condition.

Data availability

The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank the other members of the affiliation for their comments and help during the experiment. We also thank the online tool Figdraw (www.figdraw.com) for its help in the completion of Figure 8.

Funding

This work was supported by Natural Basic Research of Shaoyang (2024PT6171).

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Authors and Affiliations

  1. Department of Cardiothoracic Surgery, The People’s Hospital of Liuyang, Changsha, China

    Zhijun Fan & Zheng Yi

  2. Department of Gastrointestinal Surgery, The Central Hospital of Shaoyang, Shaoyang, China

    Sheng Li & Junjun He

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  1. Zhijun Fan
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Contributions

ZF and ZY performed the experiments and collected the data. ZF, SL and JH analyzed the data. SL prepared Figs. 1, 2, 3, 4, 5, 6 and 7. ZF wrote the main manuscript and prepared Figure 8. All authors reviewed the manuscript.

Corresponding author

Correspondence to Junjun He.

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Animal ethics declaration

All animal experiments in this study followed the ARRIVE guideline and were approved by the Ethical Committee of Hunan SJA Laboratory Animal Co., Ltd (IACUC- SJA2024017-1).

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Fan, Z., Yi, Z., Li, S. et al. Parabacteroides distasonis promotes CXCL9 secretion of tumor-associated macrophages and enhances CD8+T cell activity to trigger anti-tumor immunity against anti-PD-1 treatment in non-small cell lung cancer mice. BMC Biotechnol 25, 30 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12896-025-00963-9

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Keywords

BMC Biotechnology

ISSN: 1472-6750

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