IL-33/ST2 signalling promotes tumor growth by regulating polarization of alternatively activated macrophages

Cell culture

Mouse lung carcinoma [Lewis] (Guangzhou Cellcook Biotech Co. Ltd. Guangzhou, China), L929, and 293T cells were cultured in Dulbecco’s Modified Eagle’s Medium [DMEM] (Sartorius CellGenix GmbH, Freiburg, Germany) supplemented with 10% foetal bovine serum [FBS] (Sartorius) and 1% penicillin/streptomycin (Solarbio, Beijing, China). Bone marrow cells were removed from mice (6–8 weeks old) and cultured for 7 days in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 30% conditioned medium from the L929 cells to differentiate into bone marrow-derived macrophages (BMDMs). All cells were cultured in an incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C under 5% CO₂. All cell lines were purchased from Guangzhou Cellcook with confirmation of genotypic authentication or acquired from our laboratory.

Animals and tumor models

Il33 transgene (Il33^Tg), Il33-deficient (Il33^−/−), and Il1rl1-deficient (St2^−/−) mice were purchased from Cyagen Biosciences, Inc. (Suzhou, China). All animals were housed under specific pathogen-free (SPF) conditions in temperature-controlled, air-conditioned facilities with unlimited access to food and water. Wide-type (WT) mice were obtained from the same litter. Lewis cells (1 × 10⁶) were injected subcutaneously into the right dorsum of mice to create a mouse tumor model. Tumor volume was estimated using the following formula: tumor volume (mm³) = [length (mm) × short (mm)²]/2. Mouse models of carcinoma in situ were established, as previously described²⁴. Mice were anesthetized using an intraperitoneal injection of Avertin (200 μL [20 mg/mL] 2,2,2-tribromoethanol; Sigma-Aldrich, St. Louis, MO, USA). The lungs were then exposed surgically by making an incision (0.5 cm long) in the left chest and 2 × 10⁵ Lewis cells resuspended in 20 μL of Corning Matrigel (Corning, NY, USA) were injected into the lower left lung using a plastic, 1-mL insulin needle. The surgical wounds were closed with sutures. The mice were randomly assigned to 2 different groups 7 d later and BMDMs (1 × 10⁶ cells/mouse) derived from WT and St2^−/− mice were injected into the tail vein with repeated injections on day 10. The mice were sacrificed on day 14. All experiments were performed in accordance with the National Guidelines for Experimental Animal Welfare and were approved by the Animal Welfare and Research Ethics Committee of Jilin University (Approval no. 2022:95) prior to initiation of the study.

Human lung cancer sample and spatial transcriptomics

Surgically resected lung cancer tissues were obtained from a 61-year-old male with moderately differentiated invasive adenocarcinoma and a 71-year-old male with moderately differentiated invasive adenocarcinoma and immediately submerged in cold phosphate-buffered saline [PBS] (Bioss, Beijing, China) and cut into approximately 5 mm-thick sections. The bulk tissues were placed in an OCT-filled mould (SAKURA, Tokyo, Japan) and snap-frozen on dry ice. Spatial transcriptome sequencing based on the Visium Technology Platform from 10 × Genomics was performed by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). Written informed consent was obtained from all participants. This study was approved by the Ethics Committee of the First Hospital of Jilin University (Approval no. 2024:8).

Survival and correlation analyses

The online Kaplan–Meier plotter tool (https://kmplot.com/analysis/) was used for survival analysis. GEPIA 2 (http://gepia2.cancer-pku.cn/#index) was used for correlation analysis of ST2 expression with CD163 in the lung adenocarcinoma database.

Immunohistochemistry

Tumor tissues from mice were formalin-fixed and paraffin-embedded on slides and the paraffin-embedded tissues were de-paraffinised and rehydrated. Antigens were retrieved in a heated citrate buffer (Beyotime, Shanghai, China) for 1 h and incubated with a 3% bovine serum albumin (BSA, Sigma-Aldrich) blocking solution at room temperature for 30 min. The slides were then incubated with anti-CD31 antibody (Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. The slides were further incubated with the immunohistochemistry (IHC) detection reagent and visualised using a secondary antibody and DAB peroxidase substrate (Fuzhou Maixin Biotech. Co., Ltd, Fuzhou, China).

Immunofluorescence

Tumor tissue sections were incubated with primary antibodies (anti-ST2; R&D Systems, Minneapolis, MN, USA) and (anti-CD206; Cell Signaling Technology) at 4°C overnight, followed by incubation with the corresponding antibodies for 1 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole [DAPI] (Beyotime) for 5 min. The slides were mounted on coverslips and imaged under a microscope (Olympus, Tokyo, Japan).

Cell proliferation

BMDMs from WT and St2^−/− mice were cultured in 96-well plates for 48 h to study cellular proliferation. Lewis cells were co-cultured with BMDM-Ctrl, BMDM-flIl33, or BMDM-cIl33 cells for 48 h. The Cell Counting Kit-8 [CCK-8] (Bioss) was used to detect cell proliferation based on absorbance values following the manufacturer’s protocol.

Invasion assay

Cellular invasion was measured using Transwell inserts containing 8-μm membrane filters (LABSELECT, Jinan, China). Lewis cells (2 × 10⁴) were seeded in serum-free medium in the upper chamber of a Transwell insert coated with Matrigel (Corning). BMDM-Ctrl, BMDM-flIl33, and BMDM-cIl33 cells (1 × 10⁵) in fresh medium containing 10% FBS were seeded in the lower Transwell chamber. After incubation for 24 h, cells on the upper surface were washed with PBS, fixed with 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet for 15 min. Invasive cells were counted under a microscope.

Lentiviral packaging and overexpression assay

Lentiviral production and transduction were performed using a lentiviral vector containing a lentiviral packaging mix. The following mouse lentivirus packaging plasmids were purchased from HanBio Biotechnology Co., Ltd. (Shanghai, China): control, full-length Il33 (flIl33, 1–266 amino acids); Il33 C-terminal domain (cIl33, 109–266 amino acids); pMD2G plasmids; and psPAX2 plasmids First, the plasmids were extracted (TransGen Biotech, Beijing, China) and transfected into 293T cells using a transfection reagent (Yeasen, Shanghai, China) for 48 h. The supernatant was collected and centrifuged in ultrafiltration tubes (Merck, Darmstadt, Germany) to obtain the lentivirus. The lentivirus was transfected into Lewis cells using polybrene (Yeasen) and puromycin (Sigma-Aldrich) was added 72 h after transfection to screen the cells. After 1 week of screening, stable monoclonal cells were obtained, including Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33. To obtain BMDMs expressing flIl33 and cIl33, the lentivirus was transfected into BMDMs, BMDM-Ctrl, BMDM-flIl33, and BMDM-cIl33 cells were obtained after transfection for 72 h.

Flow cytometry analysis

Fresh tumor tissues were cut into small pieces. The tissues were dissociated using a grinder and centrifuged at 400×g for 5 min. The erythrocytes were lysed (Solarbio) and the cell suspension was incubated with antibodies. The cultured cells were then processed. Briefly, 100 μL of the cell suspension was incubated for 10 min with FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were washed with PBS to remove the unbound FcR blocking reagent. For surface staining, APC anti-mouse CD45 antibody (BioLegend, San Diego, CA, USA), PerCP anti-mouse F4/80 antibody (BD Biosciences, San Jose, CA, USA.), PE anti-mouse CD206 antibody (BD Biosciences), FITC anti-mouse CD11c antibody (BD Biosciences), PE anti-mouse NK1.1 antibody (BD Biosciences), PE anti-mouse B220 antibody (BD Biosciences), PE anti-mouse CD3 antibody (eBioscience, USA), FITC anti-mouse CD4 antibody (eBioscience, San Diego, CA, USA), PerCP-Cy5.5 anti-mouse CD8 antibody (eBioscience), APC anti-mouse CD279 (PD-1) antibody (BioLegend), and PerCP anti-mouse CTLA4 antibody (BioLegend) were added as appropriate, incubated at 4°C for 30 min, and washed with the FACS buffer (1% FBS in PBS). To stain intracellular molecules, cells were fixed with 4% paraformaldehyde for 50 min, washed twice with 0.1% saponin-PBS for permeabilisation, stained with the PE anti-mouse IFNγ antibody (eBioscience), PE anti-mouse perforin antibody (eBioscience), PE anti-mouse Foxp3 antibody (Biolegend) as appropriate for 45 min, washed with FACS buffer. All samples were analysed using a flow cytometer (Guava Technologies, Hayward, CA, USA) and analysed using FlowJo v10 (FlowJo, LLC, Ashland, OR, USA).

Macrophage phagocytosis

BMDMs from WT and St2^−/− mice were cultured in 6-well plates. Lewis cell debris with GFP fluorescence was added to the culture supernatant and incubated for 24 h. The BMDMs were collected, labelled with F480 antibody, and the percentage of F480-positive cells containing GFP was detected using flow cytometry.

Enzyme-linked immunosorbent assay (ELISA)

The cytokine concentration in the culture media from co-culture experiments and tumor tissues was quantitatively determined using an ELISA following the manufacturer’s instructions, including TGF-β (Solarbio) and IL-33 (R&D Systems). The absorbance of each well was measured at 450 nm using a microplate reader (EPOCH2, BioTek, Winooski, VT, USA).

Quantitative real-time polymerase chain reaction

Total RNA was extracted using TRIzol reagent (Takara, Kusatsu, Japan) and reverse-transcribed into cDNA using a cDNA Synthesis Kit (TOLO Biotechnology, Shanghai, China). Real-time PCR was performed using an Agilent Mx3000P machine (Agilent, Santa Clara, CA, USA) with SYBR Green Master Mix (Takara). The following forward and reverse primer sequences for the test genes were used: Gapdh (forward, 5′-GCACCGTCAAGGCTGAGAAC-3′; reverse, 5′-GGATCTCGCTCCTGGAAGATG-3′); Mrc1 (forward, 5′-CTCTGTTCAGCTATTGGACGCCG-3′); reverse, 5′-TGGCACTCCCAAACATAATTTGA-3′); Tgfb (forward, 5′-AGGCGGTGCTCGCTTTGTA-3′; reverse, 5′-CTGCTTCCCGAATGTCTGA-3′); and Arg1 (forward, 5′-CTCCAAGCCAAAGTCCTTAGAG-3′; reverse, 5′-AGGAGCTGTCATTAGGGACA-3′). The target mRNA expression was calculated relative to the amount of GAPDH using the 2^−ΔΔCt method.

Western blot analysis

The total cellular protein was collected and lysed using RIPA (Beyotime) and protease inhibitors (Roche, Basel, Switzerland) at 4°C for 30 min, centrifuged at 10,000 × g for 30 min, and the supernatant was collected. The protein concentration was determined using a BCA Protein Quantification Kit (Biomed, Beijing, China). All samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Merck). The membrane was blocked with 5% skim milk at room temperature for 2 h followed by incubation overnight at 4°C with the primary antibody against CD206 (Cell Signaling Technology), PI3K (ZenBio Science, Chengdu, China), P-PI3K (Affinity Biosciences, Changzhou, China), Akt (ZenBio Science), P-Akt (ZenBio Science), and GAPDH (Wuhan Sanying, Wuhan, China). The PVDF membranes were washed three times with Tris-buffered saline with Tween-20 [TBST] (Solarbio) for 5 min each, incubated with an HRP-conjugated secondary antibody (Bioss) for 40 min at room temperature, then washed three times with TBST. Specific bands were detected using an enhanced chemiluminescence detection reagent (Sparkjade, Jinan, China).

RNA sequencing (RNA-Seq) analysis

RNA-Seq was performed by Sangon Biotechnology (Shanghai, China). Briefly, BMDMs from WT, Il33^Tg, and St2^−/− mice were stimulated with IL-4 for 48 h and total RNA was extracted using the TRIzol Reagent (Takara). RNA integrity was assessed using an Agilent Bioanalyzer 2100. Only samples with clean rRNA profiles were used in the subsequent steps. Paired-end sequencing was performed using an Illumina HighSeq 4000 instrument (Illumina, San Diego, CA, USA).

Macrophage and Lewis cell co-culture

Macrophages and Lewis cells were co-cultured using Transwell inserts containing 0.4-μm membrane filters (LABSELECT). BMDMs from WT or St2^−/− mice were placed in the lower Transwell chamber with Lewis-NC (Lewis cells). Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 cells were added to the upper Transwell chamber and incubated for 48 h. The PI3K/Akt signalling pathway was inhibited using a PI3K-IN-1 inhibitor (catalog no. HY-12068; MCE, Monmouth Junction, NJ, USA) in the co-culture system.

T cell sorting

T cells were sorted using the EasySep™ Mouse CD8⁺ T Cell Isolation Kit and the EasySep™ Mouse CD4⁺ T Cell Isolation Kit (STEMCELL, Vancouver, Canada) following the manufacturer’s instructions. Briefly, splenocytes were resuspended in Dulbecco’s phosphate-buffered saline [DPBS] (Servicebio, Wuhan, China). One millilitre of the cell suspension was placed in a sorting tube. An FcR blocker and isolation cocktail were then added to the sample and incubated for 10 min. RapidSpheres™ were added to the samples and incubated for 5 min. Subsequently, 1.3 mL of DPBS was added to give a final volume of 2.5 mL. The sorting tube was placed in a magnet apparatus for 5 min at room temperature and the supernatants containing the cells were collected and used for further experiments.

Supernatants stimulate T cells

The BMDM supernatants co-cultured with Lewis-NC, Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 were collected and used to resuspend sorted CD4⁺ T cells and CD8⁺ T cells, which were subsequently incubated in 96-well plates for 72 h. TGF-β was neutralised using TGF-β neutralising antibody (Biolegend). The cells were collected for subsequent experiments.

Statistical analysis

All experimental results were obtained from at least three replicates. The sample size for both the animal model and in vitro experiments was decided based on expected variance and standard deviations from pilot studies. The significance was set at 0.05 with a power of 0.9 following the guidelines published in ILAR Journal²⁵. The statistical significance of differences was tested using one-way analysis of variance (ANOVA) or unpaired two-tailed t-tests. The results are presented as the mean ± standard deviation (SD). Analyses were performed using GraphPad Prism software (version 8.0; GraphPad Software, Boston, MA, USA). Statistical significance was set at a P < 0.05.

ST2 expression on macrophages is associated with lung cancer

Immunofluorescence analysis was performed on mouse lung cancer samples to determine the clinical relevance of ST2 in the context of lung adenocarcinoma (LUAD). Th ST2 expression was significantly higher in tumor tissues compared to adjacent normal lung tissues (Figure 1A). A Kaplan–Meier plot generated from an online dataset comprised of 10 independent cohorts (a total of 1161 patients) revealed that high ST2 expression correlates with reduced survival rates in patients with LUAD (Figure S1)²⁶. The detailed characteristics of each dataset are presented in Table S1. WT, Il33^Tg, Il33^−/−, and St2^−/− mice were inoculated with Lewis lung carcinoma cells to further investigate the role of IL-33/ST2 in lung cancer. The growth of tumors was faster in Il33^Tg mice (Figure 1B), while Il33 and St2 knockout inhibited tumor growth (Figure 1C). These results suggested that the IL-33/ST2 axis promotes lung cancer progression.

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Figure 1

ST2 expression on macrophages is associated with lung cancer. (A) Representative images of ST2 expression in formalin-fixed, paraffin-embedded lung cancer tissue sections from mouse (T) and adjacent normal lung tissues (N), ST2 (green), and DAPI (blue). Scale bar, 20  μm (left and right), 200 μm (middle); (B) Tumor size (left) and the change of tumor volume (right) after Lewis cells were inoculated in WT and Il33^Tg mice (n = 3); (C) Tumor size (left) and the change of tumor volume (right) after Lewis cells were inoculated in WT, Il33^−/−, and St2^−/− mice (n = 4), * WT vs. Il33^−/−, # WT vs. St2^−/−; (D) UMAP showing the distribution and clusters of ST2 in patients with lung cancer using single-cell RNA-sequencing data; (E) Correlation analysis of ST2 and CD163 genes using the database of patients with lung adenocarcinoma from the GEPIA2 website; (F) H&E staining and the spatial cluster distribution of ST2 and CD163 in tumor tissues of patients with lung adenocarcinoma. Scale bar, 2  mm. Data are shown as the mean ± SD and analysed using one-way ANOVA or an unpaired two-tailed t-test. **P < 0.01, ****P < 0.0001, ^####P < 0.0001. ST2, suppression of tumorigenicity 2; UMAP, Uniform Manifold Approximation and Projection; H&E, haematoxylin and eosin.

ST2 expression was determined in various cell types by analysing single-cell sequencing data from 49 patients with lung cancer to identify the cells in the TME with high ST2 expression²⁷. ST2 was expressed in macrophages, fibroblasts, and T cells (Figure 1D). Macrophage polarization has an important role in the TME. A correlation analysis was performed using data from the GEPIA2 website to determine the relevance of ST2 and AAMs in lung adenocarcinoma²⁸. Expression of ST2 and CD163, which are primarily expressed in AAMs²⁹, was shown to be positively correlated with LAUD (Figure 1E). Moreover, spatial transcriptomic data from biopsy tissues of patients with LUAD showed overlapping expression of ST2 and CD163 in tumor tissues (Figure 1F). These data highlighted the potential functional role of ST2⁺ macrophages in lung cancer.

Cell transfer of St2^−/− BMDMs inhibit tumor growth

St2 was knocked out and the effect on macrophage function was determined in vitro. St2 knockout did not affect the proliferative and phagocytic function of macrophages (Figure 2A, B). A mouse model of carcinoma in situ was constructed by inoculating the lungs of mice with Lewis cells to further determine whether St2 knockout macrophages (St2^−/− BMDMs) inhibit tumor growth in vivo. WT and St2^−/− BMDMs were administered via tail vein injection into mice with carcinoma in situ. The St2^−/− BMDM-treated group inhibited tumor growth (Figure 2C). Expression of CD31 in tumor tissues was assessed to determine the effect of St2^−/− BMDMs on angiogenesis. IHC results showed that St2^−/− BMDMs inhibited CD31 expression (Figure 2D). Immunofluorescence staining of ST2 and the marker of AAMs (CD206) in mouse tumor tissues showed that the St2^−/− BMDM-treated group exhibited fewer ST2⁺ AAMs than the WT BMDMs group (Figure 2E). Thus, transfer of St2^−/− BMDMs inhibited tumor growth by decreasing polarization of AAMs in the TME.

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Figure 2

Cell transfer of St2^−/− BMDMs inhibits tumor growth. (A) The effect of St2 knockout on the proliferative capacity of macrophages using CCK-8; (B) The effect of St2 knockout on the phagocytosis of macrophages using flow cytometry; (C) Tumor size (left) and tumor volume (right) after WT BMDMs and St2^−/− BMDMs were administered to the carcinoma in situ mice via the tail vein (n = 4); (D) Representative IHC images (left) and quantification analysis (right) of CD31 in tumor tissue sections after WT BMDMs and St2^−/− BMDMs were administered to the carcinoma in situ mice via the tail vein, scale bar, 100 μm (up), 20 μm (down); (E) Representative IF images (left) and quantification analysis (right) of ST2 and CD206 in formalin-fixed, paraffin-embedded sections of lung cancer tissue after WT BMDMs and St2^−/− BMDMs were administered to the mice with carcinoma in situ via the tail vein. Scale bar, 20 μm. Data are shown as the mean ± SD and analysed using an unpaired two-tailed t-test. *P < 0.05. ST2, suppression of tumorigenicity 2; BMDM, bone marrow-derived macrophages; CCK-8, Cell Counting Kit-8; IHC, immunohistochemistry; IF, immunofluorescence.

IL-33/ST2 creates an immunosuppressive TME

A lentivirus containing flIl33 and cIl33 without the nuclear localisation domain was constructed to determine the local effects of the IL-33/ST2 axis on tumor growth and eliminate the potential ST2-independent functions of flIL-33³⁰. Lewis cells overexpressing flIl33 (Lewis-flIl33), cIl33 (Lewis-cIl33), and a control (Lewis-Ctrl) were successfully generated. Lewis-Ctrl, Lewis-flIl33 and Lewis-cIl33 were implanted intradermally into WT mice to assay the effects of IL-33/ST2 in vivo. Tumor growth was significantly promoted in the Lewis-flIl33 and Lewis-cIl33 groups compared to the Lewis-Ctrl group (Figure 3A). Moreover, the ELISA results showed that IL-33 levels increased in the tumor tissues of the Lewis-flIl33 and Lewis-cIl33 groups (Figure 3B). The expression profiles of various immune cell populations were assessed using flow cytometry to determine the effect of local IL-33 on the TME. The proportion of leucocytes, macrophages, AAMs, and CD4⁺ T cells increased in the Lewis-flIl33 and Lewis-cIl33 groups, while the proportion of natural killer (NK) cells decreased compared to the Lewis-Ctrl group; the decreased proportion of CD8⁺ T cells only occurred in Lewis-cIl33 groups (Figure 3C-G, Figure S2A-E). No significant changes in other immune cells, such as dendritic cells (DC) cells, T cells, and B cells, were detected (Figure 3H-J, Figure S2F-H). Immunohistochemistry (IHC) and H&E staining were used to detect pathologic changes in the tumor tissues to further determine the effect of IL-33 on the TME. Severe necrosis and immune cell infiltration were noted in the Lewis-flIl33 and Lewis-cIl33 groups compared to the control group (Figure 3K). IHC analysis showed increased expression of CD31 in the Lewis-flIl33 and Lewis-cIl33 groups (Figure 3L). Taken together, these results suggested that IL-33/ST2 exerts a pro-tumor effect by creating an immunosuppressive microenvironment.

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Figure 3

IL-33/ST2 creates an immunosuppressive tumor microenvironment. (A) Tumor size (left) and the change in tumor volume (right) after Lewis-Ctrl (Ctrl), Lewis-flIl33 (flIl33), and Lewis-cIl33 (cIl33) cells were inoculated in WT mice (n = 6), *Lewis-Ctrl vs. Lewis-flIl33, # Lewis-Ctrl vs. Lewis-cIl33; (B) Detection of IL-33 in the tumor tissues of Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 groups using ELISA; (C-J) The proportion of immune cells in tumor tissues of Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 groups detected by flow cytometry. (C) Proportion of CD45⁺ cells in live cells; (D) Proportion of F4/80⁺ cells in CD45⁺ cells; (E) Proportion of F4/80⁺ CD206⁺ cells in CD45⁺ cells; (F) Percentage of CD4⁺ and CD8⁺ T cells in CD3⁺ cells; (G) Proportion of NK1.1⁺ cells in CD45⁺ cells; (H) Proportion of CD11C⁺ cells in CD45⁺ cells; (I) Proportion of B220⁺ cells in CD45⁺ cells; (J) Proportion of CD3⁺ cells in CD45⁺ cells; (K) Representative H&E stained images of tumor tissues from Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 groups. Scale bar, 100 μm (up), 20 μm (down); (L) Representative IHC stainedimages (left) and quantification analysis (right) of CD31 in tumor tissues from Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 groups. Scale bar, 100 μm (up), 20 μm (down). Data are shown as the mean ± SD and analysed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ^###P < 0.001. IL-33, interleukin-33; ST2, suppression of tumorigenicity 2; AAM, alternatively activated macrophages; ELISA, enzyme-linked immunosorbent assay; H&E, haematoxylin and eosin; IHC, immunohistochemistry.

IL-33 secreted by tumor cells promotes AAM polarization via ST2

IL-33 is known to promote tumor growth by increasing AAM-type macrophage infiltration via the ST2 axis in B16 melanoma²³. Lewis (Lewis-NC), Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 cells were co-cultured with WT BMDMs in vitro to determine if IL-33 influences lung cancer development by affecting macrophage polarization in the TME. The flow cytometry results showed that AAMs increased in the Lewis-flIl33 and Lewis-cIl33 groups (Figure 4A, Figure S3A). The levels of CD206 protein and Mrc1, Arg1, and Tgfb gene expression were upregulated in WT BMDMs in the Lewis-flIl33 and Lewis-cIl33 groups (Figure 4B, C). Conditioned media were collected from Lewis-NC, Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 cells, which were co-cultured with WT BMDMs, compared to media from cells that were not co-cultured to determine the secretion of IL-33. Lewis-flIl33 and Lewis-cIl33 cells secreted IL-33 with greater secretion by Lewis-cIl33 cells than Lewis-flIl33 cells. Moreover, Lewis-flIl33 and Lewis-cIl33 cells promoted secretion of IL-33 from macrophages (Figure 4D). St2^−/− BMDMs were co-cultured with Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 cells to further investigate whether macrophage polarization in the co-culture system was dependent on tumor cell-derived IL-33. Flow cytometry showed that AAMs did not increase in the Lewis-flIl33 and Lewis-cIl33 groups (Figure 4E, Figure S3B). Moreover, the levels of CD206 protein and Mrc1, Arg1, and Tgfb gene expression were not significantly altered (Figure 4F, G) and IL-33 expression did not increase in the supernatant of the co-culture system (Figure 4H). Thus, IL-33 secreted by tumor cells promoted IL-33 secretion by macrophages and promoted polarization of AAMs, which was ST2-dependent.

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Figure 4

IL-33 secreted by tumor cells promotes AAM polarization. (A-D) WT BMDMs were co-cultured with Lewis-NC (WT-NC), Lewis-Ctrl (WT-Ctrl), Lewis-flIl33 (WT-flIl33), and Lewis-cIl33 cells (WT-cIl33) for 48 h. (A) The proportion of F4/80⁺ CD206⁺ cells of WT BMDMs in co-culture systems detected using flow cytometry; (B) The protein expression of CD206 of WT BMDMs in co-culture systems detected using western blot (left) and quantification was analysed by ImageJ (right); (C) WT BMDM Mrc1, Tgfb, and Arg1 mRNA expression in co-culture systems detected using qPCR; (D) Expression of IL-33 in supernatants of Lewis-NC, Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 cells before and after culture with WT BMDMs detected using ELISA; (E-H) St2^−/− BMDMs were co-cultured with Lewis-NC (St2^−/−-NC), Lewis-Ctrl (St2^−/−-Ctrl), Lewis-flIl33 (St2^−/−-flIl33), and Lewis-cIl33 cells (St2^−/−-cIl33) for 48 h. (E) The proportion of F4/80⁺ CD206⁺ cells of St2^−/− BMDMs in co-culture systems detected using flow cytometry; (F) BMDM CD206 of St2^−/− protein expression in co-culture systems detected using western blot (left), and quantification was analysed by ImageJ (right); (G) BMDM Mrc1, Tgfb, and Arg1 of St2^−/− mRNA expression in co-culture systems detected using qPCR; (H) Expression of IL-33 in supernatants of Lewis-NC, Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 cells before and after culture with St2^−/− BMDMs detected using ELISA. Data are shown as the mean ± SD and analysed using one-way ANOVA or unpaired two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001. IL-33, interleukin-33; ST2, suppression of tumorigenicity 2; AAM, alternatively activated macrophages; BMDM, bone marrow-derived macrophages; qPCR, quantitative real-time polymerase chain reaction.

IL-33/ST2 activates PI3K-Akt during polarization of AAMs

Differentially enriched pathways in WT BMDMs, Il33^Tg BMDMs, and St2^−/− BMDMs stimulated with IL-4 were screened using transcriptome sequencing to identify the relevant IL-33/ST2 pathways in macrophage polarization. A total of 4756 differentially expressed genes were noted at the transcript level in St2^−/− BMDMs compared to WT BMDMs, 2220 differentially expressed genes were noted at the transcript level in Il33^Tg BMDMs compared to WT BMDMs, and 4215 differentially expressed genes were noted at the transcript level in St2^−/− BMDMs compared to Il33^Tg BMDMs (Figure 5A). The signalling pathways enriched for differentially expressed genes according to the KEGG database showed that the groups were enriched for the PI3K/Akt signalling pathway (Figure 5B). In addition, PI3K/Akt signalling was shown to be highly activated in macrophages compared to other immune cells in lung cancer tissues by analysing single-cell sequencing data from 49 patients with lung cancer (Figure 5C)²⁷. The levels of PI3K, p-PI3K, Akt, and p-Akt expression in WT BMDMs were measured in the co-culture system using western blot to determine whether the IL-33/ST2 axis promotes polarization of AAMs by activating the PI3K/Akt signalling pathway. Lewis-flIl33 and Lewis-cIl33 cells co-cultured with WT BMDMs showed enhanced activation of the PI3K/Akt pathway (Figure 5D, G). In contrast, no significant changes were noted in the co-culture system with St2^−/− BMDMs (Figure 5E, H). A PI3K/Akt inhibitor was added to the co-culture system to further explore whether macrophage polarization was promoted through activation of the PI3K/Akt pathway. The levels of CD206 protein were inhibited in BMDMs co-cultured with Lewis-flIl33 and Lewis-cIl33 cells after the addition of a PI3K/Akt inhibitor (Figure 5F, I). Flow cytometry results showed that AAMs were decreased in BMDMs co-cultured with Lewis-flIl33 and Lewis-cIl33 cells after the addition of a PI3K/Akt inhibitor (Figure 5J, Figure S4). Mrc1, Arg1, and Tgfb gene expression was inhibited in BMDMs co-cultured with Lewis-flIl33 and Lewis-cIl33 cells after addition of a PI3K/Akt inhibitor (Figure 5K). Overall, these results indicated that the IL-33/ST2 axis activates the PI3K/Akt signalling pathway during AAM polarization.

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Figure 5

IL-33/ST2 activates PI3K-Akt during polarization of AAMs. (A-B) RNA-seq of IL-4 stimulated BMDMs derived from WT (WT), St2^−/− (St2^−/−), and Il33^Tg mice (Il33^Tg) for 48 h; (A) Volcano plot showing gene distribution of St2^−/− vs. WT, Il33^Tg vs. WT, and St2^−/− vs. Il33^Tg. Red dots represent upregulation and green dots represent downregulation; (B) Signalling pathways for KEGG enrichment analysis of St2^−/− vs. WT, Il33^Tg vs. WT, and St2^−/− vs. Il33^Tg; (C) Signalling pathways for KEGG enrichment analysis of macrophages vs. other immune cells in lung cancer tissues by analysing single-cell sequencing data from 49 patients with lung cancer; (D) WT BMDM PI3K, p-PI3K, Akt, and p-Akt protein expression co-cultured with Lewis-NC, Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 detected using western blot; (E) St2^−/− BMDM PI3K, p-PI3K, Akt, and p-Akt protein expression co-cultured with Lewis-NC, Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 cells detected using western blot; (F) CD206 protein expression derived from WT BMDMs co-cultured with Lewis-NC, Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 cells after the addition of a PI3K/Akt inhibitor was detected using western blot; (G) Quantification of Figure 5D was analysed by ImageJ; (H) Quantification of Figure 5E was analysed by ImageJ; (I) Quantification of Figure 5F was analysed by ImageJ; (J) The proportion of F4/80⁺ CD206⁺ cells of WT BMDMs co-cultured with Lewis-NC, Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 cells after adding a PI3K/Akt inhibitor detected using flow cytometry; (K) Mrc1, Tgfb, and Arg1 mRNA expression in WT BMDMs co-cultured with Lewis-NC, Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 cells after adding a PI3K/Akt inhibitor detected using qPCR. Data are shown as the mean ± SD and analysed using an unpaired two-tailed t-test. *P < 0.05, **P < 0.01. AAM, alternatively activated macrophages; KEGG, Kyoto Encyclopedia of Genes and Genomes; BMDM, bone marrow-derived macrophages; qPCR, quantitative real-time polymerase chain reaction.

Macrophages overexpressing Il33 exerts a pro-tumor effect

Single-cell sequencing of samples was performed from 49 patients with lung cancer to determine IL-33 expression in the TME²⁷. Macrophages and fibroblasts were the cells expressing IL-33 in the human TME (Figure 6A). Lentiviruses were used to transfer flIl33 and cIl33 into the BMDMs and co-cultured them with Lewis cells to determine the effect of macrophages expressing Il33 on lung cancer in vivo. Macrophages overexpressing IL-33 promoted the proliferation and invasive ability of Lewis cells (Figure 6B, C). Macrophages overexpressing flIl33 and cIl33 were stimulated with IL-4 in vitro to further determine whether Il33-expressing macrophages influence macrophage polarization in the TME. The results of flow cytometry suggested that IL-33 promotes IL-4-induced AAM polarization (Figure 6D, Figure S5). Mrc1, Arg1, and Tgfb gene expression was upregulated in the BMDM-flIl33 and BMDM-cIl33 groups compared to the BMDM-Ctrl group (Figure 6E). These results indicated that IL-33/ST2 axis-polarized macrophages promote cancer progression.

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Figure 6

Macrophages overexpressing Il33 exerts a pro-tumor effect. (A) UMAP demonstrated the distribution and clusters of Il33 in patients with lung cancer using single-cell RNA-sequencing data; (B) The proliferation of Lewis cells after co-culture with BMDMs-Ctrl (Ctrl), BMDMs-flIl33 (flIl33), and BMDMs-cIl33 cells (cIl33); (C) Invasion of Lewis cells after co-culture with BMDM-Ctrl (Ctrl), BMDM-flIl33 (flIl33), and BMDM-cIl33 cells (cIl33). Scale bar, 100 μm; (D) Expression of AAMs in IL-4 treated BMDM-Ctrl (Ctrl), BMDM-flIl33 (flIl33), and BMDM-cIl33 cells (cIl33) was detected using flow cytometry; (E) Mrc1, Tgfb, and Arg1 mRNA expression in IL-4 treated BMDM-Ctrl, BMDM-flIl33, and BMDM-cIl33 cells detected using qPCR. Data are shown as the mean ± SD and were analysed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.01, ****P < 0.0001. UMAP, Uniform Manifold Approximation and Projection; BMDM, bone marrow-derived macrophages; qPCR, quantitative real-time polymerase chain reaction.

IL-33/ST2 axis-induced AAMs inhibit the function of T cells by TGF-β

CD4⁺ and CD8⁺ T cells were sorted from mouse spleens and cultured in the supernatant derived from BMDMs co-cultured with Lewis-NC, Lewis-Ctrl, Lewis-flIl33, and Lewis-cIl33 cells to further define the role of AAMs in the TME. The ability of CD8⁺ T cells to secrete IFN-γ and perforin was significantly inhibited by the supernatants from BMDMs co-cultured with Lewis-flIl33 and Lewis-cIl33 cells. Furthermore, PD-1 and CTLA4 expression on CD8⁺ T cells increased when cultured with BMDM supernatants co-cultured with Lewis-flIl33 and Lewis-cIl33 cells (Figure 7A, Figure S6A). Treg differentiation was also promoted in CD4⁺ T cells cultured with the BMDM supernatants co-cultured with Lewis-flIl33 and Lewis-cIl33 cells (Figure 7B, Figure S6B). The ability of CD4⁺ T cells to secrete IFN-γ was significantly inhibited when cultured with BMDM supernatants co-cultured with Leiws-flIl33 and Lewis-cIl33 cells (Figure 7C, Figure S6C). ELISA showed that BMDMs co-cultured with Lewis-flIl33 and Lewis-cIl33 groups secreted higher TGF-β compared to the Lewis-Ctrl group (Figure 7D). TGF-β neutralising antibody was added in the supernatant derived from BMDMs that had been co-cultured with Lewis-cIl33 cells to confirm that TGF-β influences T cell function. Neutralisation of TGF-β promoted the expression of CD8⁺ T cell IFN-γ and perforin (Figure 7E, Figure S6D), inhibited Treg cell differentiation, and promoted CD4⁺ T cell IFN-γ expression (Figure 7F, Figure S6E). Taken together, the results demonstrated that the IL-33/ST2 axis promoted secretion of TGF-β from AAMs, which inhibited T cell function.

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Figure 7

IL-33/ST2 axis-induced AAMs inhibit the function of T cells by TGF-β. (A) The proportion of IFN-γ, perforin, PD1, and CTLA4 in CD8⁺ T cells cultured with the supernatant from BMDMs that had been co-cultured with Lewis-NC (WT-NC), Lewis-Ctrl (WT-Ctrl), Leiws-flIl33 (WT-flIl33), and Lewis-cIl33 cells (WT-cIl33) detected using flow cytometry; (B) The proportion of Foxp3 in CD4⁺ T cells cultured with the supernatants from BMDMs that had been co-cultured with Lewis-NC, Lewis-Ctrl, Leiws-flIl33, and Lewis-cIl33 cells was detected by FCS; (C) Proportion of IFN-γ in CD4⁺ T cells cultured with the supernatant from BMDMs that had been co-cultured with Lewis-NC, Lewis-Ctrl, Leiws-flIl33, and Lewis-cIl33 cells detected using flow cytometry; (D) Level of TGF-β in the supernatant of BMDMs that had been co-cultured with Lewis-NC, Lewis-Ctrl, Leiws-flIl33, and Lewis-cIl33 cells detected using ELISA; (E-G) TGF-β neutralizing antibody was added to the supernatant derived from BMDMs that had been co-cultured with Lewis-cIl33, then cultured with T cells; (E) flow cytometry detected the proportion of IFN-γ and perforin in CD8⁺ T cells; (F) proportion of Foxp3 and IFN-γ in CD4⁺ T cells detected using flow cytometry. Data are shown as the mean ± SD and analysed using one-way ANOVA and an unpaired two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001. AAM, alternatively activated macrophages; BMDM, bone marrow-derived macrophages; TGF-β, transforming growth factor β; ELISA, enzyme-linked immunosorbent assay.