Tumor entrained neutrophils inhibit seeding in the premetastatic lung

Abstract

Primary tumors have been shown to prepare distal organs for later colonization of metastatic cells by stimulating organ-specific infiltration of bone marrow derived cells. Here we demonstrate that neutrophils accumulate in the lung prior to the arrival of metastatic cells in mouse models of breast cancer. Tumor-entrained neutrophils (TENs) inhibit metastatic seeding in the lungs by generating H(2)O(2) and tumor secreted CCL2 is a critical mediator of optimal antimetastatic entrainment of G-CSF-stimulated neutrophils. TENs are present in the peripheral blood of breast cancer patients prior to surgical resection but not in healthy individuals. Thus, whereas tumor-secreted factors contribute to tumor progression at the primary site, they concomitantly induce a neutrophil-mediated inhibitory process at the metastatic site.

Figure 1. Early transcriptional changes in the pre-metastatic lung and liver

A. Spontaneous metastatic progression from orthotopically engrafted luciferase-labeled 4T1 tumor cells to the lungs in syngeneic Balb/c mice. B. Venn diagram summarizing the transcriptional changes in the lung and the liver from sham operated vs. 4T1 tumor bearing mice (n=3 mice per group). C. Colony formation assay performed on lung and liver tissue from days 7 and 14 post tumor engraftment (n=4). D. Top 10 up-regulated genes in lung and liver expression array. Highlighted genes are expressed in myeloid cell lineages. E. Ly-6G immunohistochemistry performed on lung and liver tissues from tumor-bearing or sham-operated mice on day 7 post tumor engraftment. Graphs depicting the number of neutrophils/hpf in the lung and liver. F. Co-staining of lung tissue from tumor-bearing mice on day 7 shows that MMP9 (green) co-localizes with Ly-6G (red on upper panel) but not with F4/80 (red on lower panel). Error bars represent ± SEM. (** p<0.01). See also Figure S1.

Figure 2. Ly-6G antibody administration depletes neutrophils from the circulation, the primary tumor and the pre-metastatic lung

A. Circulating Ly-6G⁺ neutrophils in 10 days post tumor engraftment in sham-operated and tumor-bearing mice treated with control IgG or Ly-6G antibody. B. Ly-6G immunohistochemistry performed on tumors from IgG or Ly-6G antibody treated mice. C. Ly-6G immunohistochemistry performed on lung tissue from sham operated mice and tumor-bearing mice treated with control IgG or Ly-6G antibody. See also Figure S2.

Figure 3. TENs attenuate the spontaneous formation of lung metastases

A. Blood differentials showing neutrophils in control and anti-Ly-6G treated tumor-bearing mice. B. Primary tumor growth in control (IgG, n=9) and neutrophil depleted (Ly-6G, n=9) mice. C. Representative lung H&E images of control (IgG) and neutrophil depleted (Ly-6G) mice by day 25 post tumor engraftment. D. Metastatic foci size distribution in control (IgG) and neutrophil depleted (Ly-6G) mice. E. Metastatic events per lung area in control (IgG) and neutrophil depleted (Ly-6G) mice. F. Circulating Ly-6G⁺ neutrophils in tumor free (No tumor), control (IgG treated, n=5) and neutrophil depleted (anti-Ly-6G treated, n=5) MMTVPyMT/MMTV-cMyc tumor-bearing mice. G. Metastatic events per lung area in control (IgG) and neutrophil depleted (Ly-6G) mice. H. Primary tumor growth in control (IgG) and neutrophil depleted (Ly-6G) mice. Error bars represent ± SEM. See also Figure S3.

Figure 4. TENs but not G-CSF stimulated neutrophils attenuate the formation of experimental metastases

A. Representative images showing that transfer of TENs attenuates the formation of lung foci after tail-vein injection of tumor cells (n=5 per group). B. Quantification of lung specific luciferase activity shows that TENs significantly delay the formation of lung foci. C. Representative images showing that transfer of G-CSF stimulated neutrophils has no effect on the formation of lung foci (n=5 per group). D. Quantification of lung specific luciferase activity shows that G-CSF induced neutrophils do not delay the formation of lung foci. E. Multiple TENs transfers after introduction of tumor cells result in near complete ablation of lung foci formation (n=4 per group). F. Quantification of lung specific luciferase activity shows that lung foci formation in mice treated with multiple TENs transfers is ablated. Error bars represent ± SEM. (* p<0.05, ** p<0.01).

Figure 5. TENs gain a cytotoxic phenotype and kill tumor cells by initiating physical contact

A. Co-culture of 4T1 cells with neutrophils (Neut.) or a mixture of Lymphocytes and Monocytes (L&M) purified from sham-operated (Cont.) or 4T1 tumor-bearing (Tumor) mice. B. TENs in co-culture with 4T1 or MCF7 cells allowing physical contact or in a TransWell plate. C. Neutrophils purified from wild type (Cont. Neut.) or mammary tumor-bearing MMTV-PyMT mice in co-culture with 4T1 cells. D. Vascular permeability in lungs of sham operated (Cont.) and tumor-bearing (Tumor) mice. E. Neutrophils purified from healthy human volunteers (Healthy) or breast cancer patients (Patients) in co-culture with MDA-MB-231 cells. F. Time-lapse microscopy showing a co-culture of GFP labeled TENs (green) and 4T1 cells (red). G. Representative images showing (Cleaved Caspase 3 activity (NucView 488) in 4T1 cells cultured alone (top) or in co-culture with TENs (bottom). GFP intensity as an estimate for apoptosis in 4T1 cells cultured alone (No Neut.), with control neutrophils (Cont. Neut.) or TENs (Tumor Neut.). These experiments were repeated at least 3 times with similar results. Error bars represent ± SEM. (* p<0.05, ** p<0.01, ***p<0.001). See also Figure S4, Movies S1 and S2.

Figure 6. TENs cytotoxicity is mediated through the NADPH Oxidase – H₂O₂ pathway and triggered by physical contact

A. The NADPH Oxidase complex - superoxides generated by oxidizing NADPH converted into H₂O₂ by cellular superoxide dismutase (SOD). Myeloperoxidase (MPO) converts H₂O₂ to hypochlorous acid (HOCl). Apocynin inhibits the formation of the NADPH Oxidase complex, catalase catalyzes the formation of H₂O and O₂ from H₂O₂ and taurine eliminates HOCl. B. Co-culture of 4T1 tumor cells with TENs in the presence of the Apocynin, Superoxide Dismutase (SOD), or Taurine. C. H₂O₂ production in purified control neutrophils (naïve) and TENs (Tumor). D. H₂O₂ production in naïve neutrophils in the presence of PMA or vehicle control (Cont.). E. Naïve neutrophils co-cultured with 4T1 cells treated with vehicle (Cont.), or PMA allowing physical contact (PMA) or in a TransWell plate (PMA-TW). F. H₂O₂ concentration in the supernatant of TENs cultured alone (TENs) or in co-culture with 4T1 cells (TENs+4T1). G. Quantification of lung specific luciferase activity to follow metastatic progression in control (Cont. n=5) and Apocynin (Apo. n=5) treated tumor-bearing mice (Cont. n=5). These experiments were repeated at least 2 times with similar results. Error bars represent ± SEM. (* p<0.05, ** p<0.01).

Figure 7. Tumor secreted CCL2 is both required and sufficient for neutrophil entrainment

A. Neutrophils purified from naïve mice were co-cultured with 4T1 cells in the presence or absence of mCCL2, mCCL5 and rhG-CSF. B. H₂O₂ production in neutrophils from naïve mice in the presence or absence of mCCL2 and mCCL5. C. Neutrophils purified from healthy human volunteers were co-cultured with MDA-MB-231cells in the presence or absence of mCCL2, mCCL5 and rhG-CSF. D. CCL2 and CCL5 expression in 4T1 cells transduced with a non-targeting shRNA (cont.), CCL2 specific shRNA (#1 and #2), and CCL5 specific shRNA (#1 and #2). E. Co-culture of 4T1 cells with TENs purified from mice bearing control (Cont. shRNA), CCL2kd (#1 and #2) or CCL5kd (#1 and #2) tumors. F. Circulating Ly-6G⁺ neutrophils in sham operated (no tumor) and mice bearing control (Cont.) or CCL2kd tumors. G–H. Circulating levels of CCL2 and CCL5 in tumor free mice (no tumor), and in mice bearing control shRNA (Cont.) and CCL2kd (CCL2kd) tumors. I. H₂O₂ production in TENs purified from mice bearing control or CCL2kd tumors. J. Primary tumor growth in control (n=5) and CCL2kd (n=5) tumor-bearing mice. K. Lung specific luciferase activity measuring metastatic progression to the lungs in mice bearing control or CCL2kd tumors. L. Co-culture of TENs and 4T1 cells in the presence or absence of TGF-α. M. Phosphorylation of Smad2 in normal lung (Cont. lung), pre-metastatic lung (Tumor lung) and the primary tumor. These experiments were repeated at least 2 times with similar results. Error bars represent ± SEM. (* p<0.05, ** p<0.01). See also Figure S5.