Molecular mechanisms of IL-33-mediated stromal interactions in cancer metastasis

Abstract

Molecular mechanisms underlying the cancer stroma in metastasis need further exploration. Here, we discovered that cancer-associated fibroblasts (CAFs) produced high levels of IL-33 that acted on tumor-associated macrophages (TAMs), causing them to undergo the M1 to M2 transition. Genomic profiling of metastasis-related genes in the IL-33-stimulated TAMs showed a >200-fold increase of MMP9. Signaling analysis demonstrated the IL-33-ST2-NF-κB-MMP9-laminin pathway that governed tumor stroma-mediated metastasis. In mouse and human fibroblast-rich pancreatic cancers, genetic deletion of IL-33, ST2, or MMP9 markedly blocked metastasis. Pharmacological inhibition of NF-κB and MMP9 also blocked cancer metastasis. Deletion of IL-33, ST2, or MMP9 restored laminin, a key basement membrane component associated with tumor microvessels. Together, our data provide mechanistic insights on the IL-33-NF-κB-MMP9-laminin axis that mediates the CAF-TAM-committed cancer metastasis. Thus, targeting the CAF-TAM-vessel axis provides an outstanding therapeutic opportunity for cancer treatment.

Keywords: Cancer; Cell Biology; Macrophages; Oncology.

Figure 1. Noninflammatory stromal cell–derived IL-33 promotes tumor M2 TAM polarization.

(A) IL-33 protein levels in various tumor tissues (n = 3–4 samples per group). (B) IL-33 protein levels of tumor cell (GFP⁺), F4/80⁺ inflammatory cell, and F4/80^– or PDGFRβ⁺ stromal cell fractions isolated from Panc02 tumors (n = 3 samples per group). (C) Staining of Panc02 and LLC tumor tissues. H&E (scale bar: 50 μm); Masson’s trichrome (light blue, fibrotic components; scale bar: 100 μm); and PDGFRβ⁺ (red) cells and DAPI⁺ (blue) nuclei (scale bar: 20 μm). (D) Iba1⁺ (green) and CD206⁺ (red) macrophages in Panc02 tumors (scale bar: 20 μm). (E) Heatmap of subsets of M1- and M2-related genes by genome-wide expression profiling of IL-33–stimulated macrophages (n = 3 samples per group). (F) qPCR quantification of Cd206, Pdl2, and Ccr3 mRNA expression levels of IL-33–stimulated macrophages (n = 6 samples per group). NT, nontreated. (G) FACS analysis of F4/80⁺/CD206⁺, F4/80⁺/PD-L2⁺, and F4/80⁺/CCR3⁺ cell populations in IL-33–stimulated macrophages (n = 6 samples per group). (H) Schematic showing that pericyte/stromal fibroblast–derived IL-33 stimulates M2 macrophage polarization through a ST2-dependent mechanism. Mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test.

Figure 2. IL-33 upregulates MMP9 in macrophages through ST2 activation.

(A) Heatmap of a subset of cancer metastasis–related genes by genome-wide expression profiling of IL-33–stimulated macrophages (n = 3 samples per group). (B) qPCR quantification of mRNA expression levels of Mmps in IL-33–stimulated macrophages (n = 6 samples per group). (C) Inhibition of Mmp9 mRNA expression of IL-33–stimulated macrophages by a soluble ST2 (IL-33 trap) (n = 6 samples per group). sST2, soluble ST2. (D) ELISA quantification of active MMP9 protein in the conditional medium from IL-33–stimulated macrophages (n = 3 samples per group). (E) qPCR quantification of Mmp9 mRNA levels in F4/80⁺ cells isolated from Panc02 tumors grown in WT or St2^–/– mice (n = 6 samples per group). (F) Detection of MMP9 protease activity by a gelatin-based zymography assay in conditional medium derived from IL-33–stimulated macrophages in the presence or absence of a soluble ST2 (n = 3 samples per group). NT, nontreated. Mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test.

Figure 3. Transcriptional regulation of the Mmp9 promoter activity by IL-33- NF-κB signaling.

(A) Western immunoblot analysis of phosphorylation of IκBα, Erk, and p38 in vehicle- and IL-33–treated macrophages in the presence or absence of their specific inhibitors. β-Actin served as a loading control (n = 3 samples per group). WA, withaferin A; SB, SB203580. (B) qPCR quantification of Mmp9 mRNA levels of IL-33–stimulated macrophages in the presence or absence of their specific inhibitors (n = 6 samples per group). (C) Western immunoblot analysis of phosphorylation of IκBα in IL-33–stimulated macrophages in the presence or absence of a soluble ST2 (sST2). β-Actin served as a loading control (n = 3 samples per group). (D) A network scheme demonstrating protein-protein interactions linking IL-33 to NF-κB. Irak, IL-1 receptor–associated kinase; MyD88, myeloid differentiation primary response 88. (E) Immunostaining of NF-κB (green) in IL-33–stimulated macrophages at 5-, 30-, or 60-minute time points. Cell nuclei were counterstained with DAPI (red; scale bar: 25 μm). Overlapping yellow signals were randomly quantified (n = 3 fields per group). (F) Immunostaining of NF-κB (green) in IL-33–stimulated macrophages in the presence or absence of different concentrations of withaferin A. Cell nuclei were counterstained with DAPI (red; scale bar: 50 μm). Overlapping yellow signals were quantified (n = 4 random fields per group). (G) Detection of MMP9 protease activity by a gelatin-based zymography assay in conditional medium derived from IL-33–stimulated macrophages in the presence or absence of different concentrations of withaferin A (n = 4 samples per group). (H) qPCR quantification of Mmp9 mRNA levels in IL-33–stimulated macrophages in the presence or absence of different concentrations of withaferin A (n = 6 samples per group). (I) Schematic indicating 5 NF-κB–binding sites located in the proximal region of the mouse Mmp9 promoter. (J) Mmp9 promoter activity in macrophages by the luciferase reporter assay. Relative luciferase activity was measured after IL-33 stimulation in the presence or absence of different concentrations of withaferin A (n = 3 samples per group). (K) Schematic showing the IL-33-ST2-NF-κB-MMP9 signaling pathway. IKK, IκB kinase. Mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test.

Figure 4. IL-33-ST2–dependent production of MMP9 in macrophages.

(A) Immunohistochemical staining and quantification of Iba1⁺ (red) and CD206⁺ (green) macrophages in Panc02 tumors grown in WT, Il33^–/–, or St2^–/– mice (n = 8 random fields per group; scale bar: 100 μm). (B) Immunohistochemical staining and quantification of Iba1⁺ (red) and CD206⁺ (green) macrophages in vehicle- and soluble ST2–treated (sST2-treated) Panc02 tumors (n = 8 random fields per group; scale bar: 100 μm). (C) qPCR quantification of Mmp9 mRNA levels in Panc02 tumors grown in WT, Il33^–/–, or St2^–/– mice or WT mice treated with a soluble ST2 (n = 6 samples per group). (D) qPCR quantification of Mmp9 mRNA levels of PBS- and clodronate-treated Panc02 tumors (n = 6 samples per group). (E) qPCR quantification of Mmp9 mRNA levels in Panc02 tumors treated with vehicle and DHMEQ (a NF-κB inhibitor) (n = 6 samples per group). (F) Detection of MMP9 protease activity by a gelatin-based zymography assay in PBS- and clodronate-treated Panc02 tumors (n = 4 samples per group). (G) ELISA quantification of active MMP9 protein in PBS- and clodronate-treated Panc02 tumors (n = 3 samples per group). Mean ± SEM. **P < 0.01; ***P < 0.001, Student’s t test.

Figure 5. Activation of IL-33-ST2-NF-κB-MMP9 signaling promotes metastasis.

(A) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing WT, Il33^–/–, or St2^–/– mice. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule (T) and surrounding lung tissues (L) (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis (n = 6–10 mice per group). (B) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing mice receiving vehicle or soluble ST2 (sST2) treatments. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis (n = 6–10 mice per group). (C) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing mice receiving PBS or clodronate treatments. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis (n = 6–10 mice per group). (D) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing mice receiving vehicle or DHMEQ (a NF-κB inhibitor) treatments. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis (n = 6–10 mice per group). (E) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing mice receiving vehicle or SB-3CT (a MMP9 inhibitor) treatments. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis (n = 6–10 mice per group). (F) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing WT or Mmp9^–/– mice. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of percentage of animals with visible pulmonary metastasis (n = 6–10 mice per group).

Figure 6. Degradation of the basement membrane by the IL-33-ST2-NF-κB-MMP9 axis.

(A) Immunohistochemical staining and quantification of CD31⁺ (red) and laminin⁺ (green) structures in Panc02 tumors grown in WT, Il33^–/–, or St2^–/– mice. Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin⁺/CD31⁺ signals per field (n = 8 random fields per group; scale bar: 100 μm). (B) Immunohistochemical staining and quantification of CD31⁺ (red) and laminin⁺ (green) structures in vehicle- or soluble ST2–treated Panc02 tumors Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin⁺/CD31⁺ signals per field (n = 8 random fields per group; scale bar: 100 μm). (C) Immunohistochemical staining and quantification of CD31⁺ (red) and laminin⁺ (green) structures in PBS- or clodronate-treated Panc02 tumors. Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin⁺/CD31⁺ signals per field (n = 8 random fields per group; scale bar: 100 μm). (D) Immunohistochemical staining and quantification of CD31⁺ (red) and laminin⁺ (green) structures in Panc02 tumors treated with vehicle or DHMEQ (a NF-κB inhibitor). Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin⁺/CD31⁺ signals per field (n = 8 random fields per group; scale bar: 100 μm). (E) Immunohistochemical staining and quantification of CD31⁺ (red) and laminin⁺ (green) structures in Panc02 tumors treated with vehicle or SB-3CT (a MMP9 inhibitor). Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin⁺/CD31⁺ signals per field (n = 8 random fields per group; scale bar: 100 μm). (F) Immunohistochemical staining and quantification of CD31⁺ (red) and laminin⁺ (green) structures in Panc02 tumors grown in WT or Mmp9^–/– mice. Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin⁺/CD31⁺ signals per field (n = 8 random fields per group; scale bar: 100 μm). Mean ± SEM. **P < 0.01; ***P < 0.001, Student’s t test.

Figure 7. IL-33–induced MMP9 in macrophages promotes metastasis in human PDAC.

(A) IL-33 protein levels in human melanoma and pancreatic cancers (n = 3 samples per group). (B) Staining of MiaPaCa2 PDAC and UACC257 melanoma tumor tissues. Light blue indicates fibrotic components (Masson’s trichrome; scale bar: 50 μm). (C) qPCR quantification of Mmp9 mRNA expression in 3 human pancreatic tumors (n = 6 samples per group). (D) Immunohistochemical staining and quantification of Iba1⁺ (red) macrophages in vehicle- and a soluble ST2–treated MiaPaCa2 tumors (n = 8 random fields per group; scale bar: 100 μm). (E) Immunohistochemical staining and quantification of CD31⁺ (red) and laminin⁺ (green) structures in vehicle- and a soluble ST2–treated MiaPaCa2 tumors. Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin⁺/CD31⁺ signals per field (n = 8 random fields per group; scale bar: 100 μm). (F) Immunohistochemical staining and quantification of CD31⁺ (red) and laminin⁺ (green) structures in vehicle- and SB-3CT–treated MiaPaCa2 tumors. Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin⁺/CD31⁺ signals per field (n = 8 random fields per group; scale bar: 100 μm). (G) Lung histology of MiaPaCa2 tumor-bearing mice receiving vehicle or a soluble ST2 treatments. Dashed line marks the border between the metastatic nodule (T) and surrounding lung tissues (L) (scale bar: 250 μm). Quantification of percentage of animals with visible pulmonary metastasis (n = 6–10 mice per group). (H) Lung histology of MiaPaCa2 tumor-bearing mice receiving vehicle or SB-3CT treatments. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis (n = 6–10 mice per group). Mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test.

Figure 8. Mechanisms of IL-33–mediated crosstalk between CAFs and TAMs in cancer metastasis.

Mechanistic insights on IL-33–mediated crosstalk between pericytes (PCs)/cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) in cancer metastasis. PDGFRβ⁺ PCs/CAFs produce high levels of IL-33, which recruits TAMs and induces M2 macrophage polarization through its ST2 receptor. IL-33–stimulated TAMs produce high levels of MMP9 through activation of NF-κB, which transcriptionally activates the MMP9 promoter. MMP9 in turn degrades laminin, one of the key components in the basement membrane (BM). Deterioration of BM around the tumor vasculature allows tumor cells (TCs) to intravasate into the circulation. Circulating tumor cells (CTCs) eventually recolonize in distal organs, such as lungs, for further growth to become the clinically detectable metastatic mass.