Tumors exploit CXCR4^hiCD62L^lo aged neutrophils to facilitate metastatic spread

Figures & data

Figure 1. CXCR4^hiCD62L^lo aged neutrophils accumulate in metastatic cancer.

(a) Gating strategy for the identification of CXCR4^hiCD62L^lo aged neutrophils in mice with or without metastatic tumor cells. (b) Flow cytometric assessment of CXCR4^hiCD62L^lo neutrophil proportions in blood, lung, and liver of WT (Balb/C) compared to mice inoculated with 4T1 for 14 d. n = 5 per group. (c) Proportions of aged neutrophil in blood, lung, and liver of WT (C57BL6/J) and mice inoculated with B16F10, B16F0, or B16LS9 tumor cells for 14 d. n = 5 per group. (d,e) Number of liver metastasis of in mice treated with or without antibiotics. (d) shows 4T1 metastasis, while (e) indicates B16LS9 metastasis. Gut microbiota was depleted for 14 d before tumor inoculation. n = 7 per treatment group. (f) Strategy for treatment with antibiotic for 14 d to deplete aged neutrophils followed by i.v. adoptive transfer of naïve or aged neutrophils with tumor inoculation. (g) Number of liver metastasis by 4T1 (left) or B16LS9 (right) in antibiotic-treated mice receiving PBS, naïve or aged neutrophils. Data plotted as mean ± SEM. Each symbol represents result from each mouse. ns = not significant, *p < .05, **p < .01 by t test except C,G (ANOVA).

Figure 2. Metastatic cancers increase neutrophil aging

(a,b) Assessment of circadian fluctuations in CXCR4^hiCD62L^lo aged neutrophil numbers in the circulation at different times of the day (ZT0-ZT23). (a) represents BALB/c strain with or without syngeneic 4T1 tumor, and (b) shows n= 10 per ZT time point per experimental group. (c,d) Analysis of in vitro neutrophil aging during co-culture of bone marrow neutrophils with control media or tumor-conditioned media. In (c), Balb/C cells are co-cultured with 4T1-conditioned media. In (d), C57BL6/J cells are co-cultured with control media or B16F0, B16F10, or B16LS9-conditioned media. Experiments were performed three independent times. (e) In vitro neutrophil aging by stimulation with G-CSF (100 ng/ml), GM-CSF (100 ng/ml), or angiotensin (Ang) II (100 µM) for 3 h. (f) Effect of Ang II inhibition on neutrophil aging. Cells were treated with normal media or conditioned media from 4T1 or B16LS9 tumors. Experiments were performed for 3 hrs in the presence or absence of 1 uM of losartan (Los). (g) Schema for the adoptive transfer of naïve neutrophils isolated from GFP mice into WT or 4T1-tumor-bearing mice. (h) Aging of the donor GFP cells were then assessed by flow cytometry. Recipient mice were untreated WT, or 4T1 mice with or without hydralazine or losartan. n = 3–4 recipients per group. Each symbol represents result from one mouse. For cell cultures, cells from each mouse (three mice per experiment) were seeded in triplicates for each condition. Data plotted as mean ± SEM. ns not significant, *p < .05, **p < .01 by ANOVA, except F (t test).

Figure 3. Characteristics of aged neutrophils

Measurement of (a) neutrophil extracellular traps (NETs), (b) ROS, (c) matrix-metalloproteinase 9 (MMP-9), and (d) and neutrophil elastase from naïve and aged neutrophils obtained from 4T1 (blue) and B16F10 (pink) tumor-bearing mice. n = 4–5 mice per tumor condition. Flow cytometric assessment of the expression of occludin-1 (e) and VE-cadherin (f) by HUVEC cells cultured in the presence of naïve or aged neutrophils. (g) Representative images and quantitation of migration of tumor cells over a scratch wound at 0, 16, and 24 h. Tumor cells (4T1) were cultured with control media, naïve neutrophil media, or aged neutrophil media. Experiment was performed two independent times. (h) Measurement of tumor proliferation during co-culture with normal media or media conditioned from aged or naïve neutrophils. Cells were cultured up to 72 h. NS not significant, *p < .05, **p < .01 by unpaired Student’s t test. Data are plotted as mean ± SEM.

Figure 4. CXCR4^hiCD62L^lo aged neutrophils promote tumor metastasis via NETs

(a) Experimental design for the transfer of aged neutrophils and treatment with specific inhibitors after tumor inoculation. (b) Effect of MMP9 inhibitor (sivelestat, 50 mg/kg), neutrophil elastase inhibitor (SB-3 CT, 50 mg/kg) and NETs inhibitor (DNase I, 50 µg) on 4T1 tumor metastasis to the liver. n = 5 mice per treatment group. (c) Effect of sivelestat (50 mg/kg), SB-3 CT (50 mg/kg), and DNase I (50 µg) on B16LS9 metastasis to the liver. n = 6 mice per treatment group. ns = not significant. *p < .05, **p < .01, ***p<.001 by Tukey test. Data are plotted as mean ± SEM. (d) Proposed mechanism for diversified approaches used by cancer cells to utilize granulocytes to promote metastasis.