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. 2017 Jan 26;36(4):482-490.
doi: 10.1038/onc.2016.217. Epub 2016 Jul 4.

TNFα-activated mesenchymal stromal cells promote breast cancer metastasis by recruiting CXCR2+ neutrophils

P F Yu  1 , Y Huang  1 , Y Y Han  1 , L Y Lin  1 , W H Sun  1 , A B Rabson  2 , Y Wang  1 , Y F Shi  1   2   3
Affiliations

TNFα-activated mesenchymal stromal cells promote breast cancer metastasis by recruiting CXCR2+ neutrophils

P F Yu et al. Oncogene. .

Abstract

Mesenchymal stromal cells (MSCs) tend to infiltrate into tumors and form a major component of the tumor microenvironment. Our previous work demonstrated that tumor necrosis factor α (TNFα)-activated MSCs significantly promoted tumor growth. However, the role of TNFα-treated MSCs in tumor metastasis remains elusive. Employing a lung metastasis model of murine breast cancer, we found that TNFα-activated MSCs strikingly enhanced tumor metastasis compared with normal MSCs. We analyzed the chemokine profiles and found that the expression of CCL5, CCR2 and CXCR2 ligands were enhanced in TNFα-activated MSCs. Using genetic or pharmacological strategies to inhibit CCL5 or CCR2, we demonstrated that CCL5 and CCR2 ligands were indispensable in supporting TNFα-activated MSCs to promote tumor metastasis. Analysis of immune cells revealed that CXCR2 ligands (CXCL1, CXCL 2 and CXCL5) expressed by TNFα-activated MSCs efficiently recruited CXCR2+ neutrophils into tumor. These neutrophils were responsible for the pro-metastatic effect of MSCs since inhibition of this chemotaxis abolished increased neutrophil recruitment and tumor metastasis. The interaction between neutrophils and tumor cells resulted in markedly elevated metastasis-related genes by tumor cells, including CXCR4, CXCR7, MMP12, MMP13, IL-6 and TGFβ. Importantly, in IL8high human breast cancer samples, we also observed similar alterations of gene expression. Collectively, our findings demonstrate that TNFα-activated MSCs promote tumor metastasis via CXCR2+ neutrophil recruitment.

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Figures

Figure 1
Figure 1
TNFα-activated MSCs promote tumor metastasis. (a) 4T1 breast cancer cells (4 × 105) were co-injected with MSCs (1 × 105) into the fat pad of BALB/c mice; the control group received 4T1 cells alone. MSCs were activated by TNFα (10 ng/ml) for 24 h. Mice were euthanized after 4 weeks. Metastatic tumor nodules in the lung were counted. (b) Animal survival analysis. BALB/c mice received 4T1 cells alone or with different MSCs as described in a. (c) 4T1 breast cancer cells (4 × 105) were injected into fat pad of BALB/c mice. Mice were euthanized after 14 days. The concentration of TNFα in serum was tested by ELISA assay. TNFα levels in serum of normal mice served as a control. (d) Animal model was as described in a. Metastatic tumor nodules in the lung were counted. (e and f) For the wound-healing assay, cultured 4T1 cells were first starved for 24 h. Wound closures were photographed (e) and statistically analyzed (f) at 24 h after wounding (scale bars: 700 μm). Data are shown as means±s.e.m. Statistical significance was assessed by unpaired, two-tailed Student's t-test.
Figure 2
Figure 2
TNFα-activated MSCs promote tumor metastasis did not through CCL5 and CCR2 ligands. (a) Data sets of chemokine mRNA expression by MSCs were obtained through qPCR array. Only the chemokines with more than fivefold changes are shown. (b and c) Animal experiments were as described in Figure 1a. CCL5-KO MSCs were activated by TNFα (10 ng/ml) for 24 h. Metastasis lung tumor nodules were identified by whole-lung images (stained by bouin's solution), H&E staining (b) (scale bars: 500 μm) and counted (c). (d) BALB/c mice received 4T1 cells alone or with different MSCs as described in Figure 1a. Mice were euthanized after 10 days. Single-cell suspensions prepared from tumor tissues were analyzed for the frequency of CD11b+ F4/80+ macrophages by flow cytometry. (e and f) Animal experiments were as described in Figure 1a. RS504393 was injected intraperitoneally (2 mg/kg) every day. Tumors were excised after 4 weeks. Metastatic tumor nodules in the lung were detected by whole-lung images (stained by bouin's solution) (e) and counted (f). Data are shown as means±s.e.m. Statistical significance was assessed by unpaired, two-tailed Student's t-test.
Figure 3
Figure 3
TNFα-activated MSCs recruit more neutrophils to tumor. (a and b) Peripheral blood cells from 4T1 tumor-bearing mice were stained for CD11b, Ly6G, CXCR2 and other immune cell markers and analyzed by flow cytometry. (c and d) BALB/c mice received 4T1 cells alone or with different MSCs as described in Figure 1a. Mice were euthanized after 10 days. Single-cell suspensions prepared from tumor tissues (c and d) or from peripheral blood (c) were analyzed for the frequency of CD11b+ Ly6G+ neutrophils by flow cytometry. Data are shown as means±s.e.m. Statistical significance was assessed by unpaired, two-tailed Student's t-test.
Figure 4
Figure 4
TNFα-activated MSCs secrete CXCR2 ligands and recruit neutrophils. (a) MSCs were generated from primary tumor, bone marrow and lung of the mice bearing 4T1 tumor. Expression of chemokines in MSCs was determined by qPCR at passage 1. (b) MSCs and 4T1 cells were cultured in vitro and activated by TNFα (10 ng/ml) for 12 h. Chemokine expression was determined by qPCR. (c) MSCs were cultured in vitro and activated by TNFα (10 ng/ml) for 24 h. Chemokine levels in the medium were determined by ELISA assay. (d) In the transwell assay, neutrophils were seeded in the top well and MSC-cultured medium (MSC-CM) placed in the bottom well. SB265610 was added to both the top and bottom wells. Data are shown as means±s.e.m. Statistical significance was assessed by unpaired, two-tailed Student's t-test.
Figure 5
Figure 5
Neutrophils promote tumor metastasis. (a and b) For the wound-healing assay, cultured 4T1 cells were first starved for 24 h. New medium was added and freshly isolated neutrophils were added. Wound closures were photographed (a) and statistically analyzed (b) at 24 h after wounding (scale bars: 700 μm). (c) 4T1 breast cancer cells (4 × 105) alone or with freshly isolated neutrophils (4 × 105) were co-injected into fat pad of BALB/c mice. Mice were euthanized after 4 weeks. Metastasis of tumor nodules on lung was displayed by counting. (d and e) Animal experiments were as described in Figure 1a. SB265610 was i.p injected (2 mg/kg) to mice every days. Metastasis of tumor nodules on lung was displayed by counting (d), HE staining (scale bars: 700 μm) and whole-lung images (stained by bouin's solution) (e). (f) Animal survival analysis. BALB/c mice were injected with 4T1 cells alone or with different MSCs as described in Figure 1a. SB265610 was i.p injected (2 mg/kg) to mice every 2 days. Data are shown as means±s.e.m. Statistical significance was assessed by unpaired, two-tailed Student's t-test.
Figure 6
Figure 6
Neutrophils induce expression of pro-metastatic factors in tumor cells. (a) 4T1 cells were co-cultured with freshly isolated neutrophils for 12 h, after which time, the neutrophils were washed off of the plate and gene expression in 4T1 cells was detected by qPCR. (b) mRNA levels of S100A8/9 in different cells were detected by qPCR. (c and d) Overall survival (c) and distant metastasis-free survival (d) of breast cancer patients with high- or low- IL8 (Affymetrix ID: 202859_x_at) expression. Hazard ratios (HR) and P-values (log-rank P) were depicted for each survival analysis. Kaplan–Meier survival data were generated using the publicly online tool KM-plotter. (e) IL8 expression in TNFα-treated human MSCs (10 ng/ml, 12 h) was analyzed by real-time PCR. (f) Correlation analysis of IL8 and other pro-metastatic genes expression in breast cancer patient samples.
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