Eosinophilic inflammation promotes CCL6-dependent metastatic tumor growth

Abstract

Compelling evidence suggests that inflammatory components contribute to cancer development. However, eosinophils, involved in several inflammatory diseases, were not fully explored in cancer metastasis. We show that airway inflammatory eosinophilia and colonic inflammation with eosinophil infiltration are both associated with increased metastasis in mice. Eosinophilia is responsible for increased bone metastasis in eosinophil-enriched Cd3δ-Il-5 transgenic (Il-5 Tg) mice. We also observe increased eosinophils in the malignant pleural effusion of cancer patients with pleural metastasis. Mechanistically, eosinophils promote tumor cell migration and metastasis formation through secreting C-C motif chemokine ligand 6 (CCL6). Genetic knockout of Ccl6 in Il-5 Tg mice remarkably attenuates bone metastasis. Moreover, inhibition of C-C chemokine receptor 1 (CCR1, the receptor of CCL6) in tumor cells reduces tumor cell migration and metastasis. Thus, our study identifies a CCL6-dependent prometastatic activity of eosinophils, which can be inhibited by targeting CCR1 and represent an approach to preventing metastatic disease.

Fig. 1. Airway inflammation and colonic inflammation promotes metastasis.

(A) Schematic timeline representing the merger of an established model of allergic airway inflammation and the metastasis of circulating B16-F10 cells to the lung. (B) Representative photograph of pulmonary metastatic foci on day 42. Scale bar, 5 mm. (C) The number of experimental pulmonary metastases. (D) Schematic timeline representing the merger of an established model of colitis and the metastasis of abdominal B16-F10 cells to the colon. (E) Representative photograph of colonic metastatic foci produced after injection of B16-F10 cells on day 22. Scale bar, 1 cm. (F) The number of colon metastases. (G) The number of experimental pulmonary metastases from mice treated with NS, dexamethasone (DEX), OVA, and OVA and DEX (OVA + DEX), respectively. (H) Representative lung hematoxylin and eosin section of metastases. Scale bars, 500 μm. (I) Quantification of the tumor burden per lung (mm²) in (H). (C, F, G, and I) n = 5 to 7 mice per group. Statistical analyses were performed by Student’s t test (C and F) and one-way ANOVA (G and I). Data are presented as mean ± SEM from one representative experiment of three independent experiments. **P < 0.01; ***P < 0.001.

Fig. 2. Eosinophilia mediates enhanced bone metastasis in Il-5 Tg mice.

(A) The number of eosinophils in BM of WT and Il-5 Tg mice, n = 4 mice per group. (B) Comparison of metastasis foci of spines on day 14. WT and Il-5 Tg mice were injected with B16-F10 cells via caudal arteries. (C) The proportion of metastasis in the spine from mice injected with B16-F10 intravenously (WT, n = 20; Il-5 Tg, n = 26). (D) Representative photograph of metastatic foci in the spine of WT and Il-5 Tg mice injected with B16-F10 intravenously on day 14. (E) The number of the spine metastasis in (D). (F) Schematic representation of BM transplantation followed by establishing a metastasis model. (G) Frequencies of eosinophils in BM of WT mice that have received donor BM transplants from WT, Il-5 Tg, or Il-5 Tg and Eos-null mice 28 days ago. (H) Comparison of the metastatic foci in the spine on day 42. (I) Quantification of the tumor burden per area (mm²) in the spine on day 42. (B, E, and G to I) n = 6 to 8 mice per group. Statistical analyses were performed by Student’s t test (A, B, and E), one-way ANOVA (G, H, and I), and Fisher’s exact test (C). Data (mean ± SEM) are from one representative experiment of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 3. Eosinophils promote tumor cell migration and metastasis formation.

(A) Schematic diagram, representative crystal violet staining, and quantification of migrated B16-F10 cells cocultured with eosinophils. (B) Schematic diagram, representative crystal violet staining, and quantification of migrated B16-F10 cells toward eosinophils in the lower compartment. (C) Schematic diagram, representative crystal violet staining, and quantification of migrated B16-F10 cells toward BM supernatants from WT or Il-5 Tg mice. (D) Comparison of the metastatic foci in the lung on day 14. Mice were injected with tumor cells and eosinophils (Eos) or only tumor cells (PBS) intravenously (i.v.). (E) Comparison of the metastatic foci in the lung on day 14. Mice were injected with tumor cells (intravenously) and immediately transferred of eosinophils or PBS [intratracheally (i.t.)] into the lung. (D) and (E) show the representative photograph and quantification of lung metastasis on day 14, n = 6 mice per group. Statistical analyses were performed by Student’s t test (A to E). Data are presented as mean ± SEM from one representative experiment of three independent experiments. *P < 0.05 and ***P < 0.001.

Fig. 4. CCL6 mediates eosinophil-induced tumor cell migration and metastasis formation.

(A) Representative images of control pleural effusion cells from non–cancer patients and MPE cells from patients with cancer with pleural metastasis. The left panel is Wright-Giemsa staining. Scale bars, 50 μm. Arrows, eosinophils. The right panel is EPX staining. (B) The number of eosinophils in control pleural effusion (n = 8) and MPE (n = 8) by flow cytometry analysis. (C) The ratio of a panel of cytokine and chemokine mRNA in WT BM cells versus Il-5 Tg BM cells, as assayed by RT-qPCR. (D) The protein levels of CCL23 in pleural fluid from patients with pleural metastasis (n = 8) and non–cancer patients (n = 8). (E) The protein levels of CCL6 in control medium (CM), the culture supernatant from eosinophils (Eos), neutrophils (Neu), and bone marrow–derived macrophages (BMDM). (F) The number of migrated B16-F10 cells per field toward CCL6-sufficient eosinophils (Eos, isolated from Il-5 Tg mice) or CCL6-deficient eosinophils (Eos^Ccl6−/−, isolated from Il-5 Tg and Ccl6^−/− mice). (G and H) Representative photograph and quantification of metastatic foci in lungs on day 14. WT mice were injected with B16-F10 cells (intravenously) and transferred with Eos or Eos^Ccl6−/− (intratracheally). Scale bar, 5 mm. (I and J) Representative photograph and quantification of metastatic foci in the spine on day 14. WT, Ccl6^−/−, Il-5 Tg, and Il-5 Tg and Ccl6^−/− mice were injected with B16-F10 cells via caudal arteries. Scale bar, 5 mm. (H and J) n = 5 to 6 mice per group. Statistical analyses were performed by Student’s t test (B to F) and one-way ANOVA (H to J). Data are presented as mean ± SEM from one representative experiment of three independent experiments (C, E, F, H, and J) or represented as box; the whiskers indicate Tukey (B and D). *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5. CCR1 is required for eosinophilia-dependent metastasis.

(A) The number of migrated CCR1-deficient B16-F10 cells (shCCR1 B16-F10) and CCR1-sufficient B16-F10 cells (shCon B16-F10) from the upper compartment toward eosinophils in the lower compartment of the Transwell system. (B) The number of metastatic foci in the spine on day 14. WT and Il-5 Tg mice were injected with CCR1-deficient B16-F10 cells or CCR1-sufficient B16-F10 cells via caudal arteries, n = 7 to 10 mice per group. (C) The number of lung metastatic foci on day 42 according to Fig. 1A. NS or OVA-challenged WT mice were injected with CCR1-deficient B16-F10 cells or control B16-F10 cells (intravenously), n = 7 to 8 mice per group. (D and E) Representative crystal violet staining and quantification of migrated B16-F10 cells in the Oris cell migration assay. B16-F10 cells were cultured in control medium or eosinophil culture supernatant after migration for 36 hours treated with or without 20 μM BX471. (F) Representative photograph of metastasis foci in the spine of Il-5 Tg mice injected with DMSO or BX471 pretreated tumor cells via caudal arteries. Scale bar, 5 mm. (G) Quantification of metastasis foci in the spine of WT and Il-5 Tg mice after injection of DMSO or BX471 pretreated tumor cells via caudal arteries. WT, n = 9 to 10 mice per group; Il-5 Tg, n = 5 mice per group. (H) Schematic representation of the proposed mechanism underlying eosinophilia-dependent metastasis. Statistical analyses were performed by one-way ANOVA (B and D). Data are presented as mean ± SEM from one representative experiment of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.