Asthma-related inflammation promotes lung metastasis of breast cancer cells through CCL11-CCR3 pathway

Abstract

Background: Mechanisms that preclude lung metastasis are still barely understood. The possible consequences of allergic airways inflammation on cancer dissemination were studied in a mouse model of breast cancer.

Methods: Balb/c mice were immunized and daily exposed to ovalbumin (OVA) from day 21. They were subcutaneously injected with 4T1 mammary tumor cells on day 45 and sacrificed on day 67. Lung metastases were measured by biophotonic imaging (IVIS® 200 Imaging System) and histological measurement of tumor area (Cytomine software). Effects of CCL11 were assessed in vivo by intratracheal instillations of recCCL11 and in vitro using Boyden chambers. CCR3 expression on cell surface was assessed by flow cytometry.

Results: The extent of tumor metastases was significantly higher in lungs of OVA-exposed mice and increased levels of CCL11 expression were measured after OVA exposure. Migration of 4T1 cells and neutrophils was stimulated in vitro and in vivo by recCCL11. 4T1 cells and neutrophils express CCR3 as shown by flow cytometry and a selective CCR3 antagonist (SB-297006) inhibited the induction of 4T1 cells migration and proliferation in response to recCCL11.

Conclusions: Allergic inflammation generated by exposure to allergens triggers the implantation of metastatic cells from primary breast tumor into lung tissues plausibly in a CCL11-CCR3-dependent manner. This indicates that asthma related inflammation in lungs might be a risk factor for lung metastasis in breast cancer patients.

Keywords: Asthma; Breast cancer; CCL11–CCR3; Lung inflammation; Lung metastasis.

Fig. 1

Inflammation generated by OVA challenge induces tumor cell dissemination to the lung parenchyma (n = 8/group). a Timeline of mice challenged with OVA and s.c injected with 4T1 cells. b Volume of subcutaneous primary tumor. c, d Tumor size quantification assessed by measuring the ratio between the area of tumor foci in lungs and total lung tissue area on 8 sections per mouse in each group. Scale bar: 2 mm (hematoxylin–eosin). Results are expressed as mean tumor area/lung area ± SEM and are representative of 2 individual experiments. OVA indicate ovalbumine; PBS, phosphate-buffered saline (e) representative BALF total cell of mice exposed to OVA and subcutaneously injected with 4T1 cells. Scale bar: 50 µm (hematoxylin–eosin). f Quantification of isolated neutrophils migrating to a gradient of recCCL11 during 4 h using Boyden chamber assay. Neutrophils migration was estimated on 16 random fields (20 ×) of triplicate wells.*p < 0.05

Fig. 2

Intratracheal injection of CCL11 recombinant (recCCL11) induces 4T1 tumor cell migration to lung tissue (n = 6/group). a Timeline of experiment. Mice were challenged with i.t of recCCL11 and i.v injected with luciferase-stably transfected 4T1 cells. b, c On day 15, biophotonic monitoring of lung metastasis in animals and quantification of bioluminescence in regions of interest (ROI) determined around lungs. d, e Representative hematoxylin–eosin–stained sections of lung tissues and tumor size quantification assessed by measuring the ratio between the area of tumor foci in lungs and total lung tissue area on 8 sections per mouse in each group. Scale bar: 2.5 mm, 100 µm. Results are expressed as mean tumor area/lung area ± SEM. **p < 0.01

Fig. 3

Measurement of CCR3 on 4T1 cells and neutrophils by flow cytometry. a The dot plot diagrams represent typical apoptotic and necrotic 4T1 cell populations detected by Annexin V-FITC and PI staining. The lower left quadrants (Q3; 49.2%) of the panels show viable intact cells, which were negative for Annexin V-FITC binding and excluded PI staining (FITC-/PI-); the upper right quadrants (Q1; 7.5%, Q2; 32.1%) show nonviable, necrotic cells, which were positive for Annexin V-FITC binding and PI uptake (FITC + /PI +). The lower right quadrants (Q4; 11.2%) represent apoptotic cells, positive for Annexin V-FITC and negative for PI (FITC + /PI-). b Representative histograms for flow cytometric analysis of surface expression of CCR3 receptors on 4T1 cells. IgG: Isotype control. c Lung neutrophils of mice treated with intratracheal instillation of recCCL11, sorted by FACS Aria

Fig. 4

Measurement of CCR3 antagonist (SB-297006) effects on 4T1 cells migration and proliferation. a Quantification of 4T1 tumor cells chemotaxis, using Boyden chamber assay, supplemented with recCCL11 and CCR3 antagonist (FBS1%: n = 8. FBS 1% + RecCCL11: n = 6 and FBS 1% + RecCCL11 + SB: n = 5), after 16 h. b After 72 h, proliferation of 4T1 cells treated with recCCL11 and CCR3 antagonist was assessed using BrdU staining (FBS 1%: n = 16, FBS 1% + RecCCL11: n = 14 and FBS 1% + RecCCL11 + SB: n = 14). Results are expressed as mean ± SEM. *p < 0.05, ***p < 0.001