Abrogation of TGF beta signaling in mammary carcinomas recruits Gr-1+CD11b+ myeloid cells that promote metastasis

Abstract

Aberrant TGFbeta signaling is common in human cancers and contributes to tumor metastasis. Here, we demonstrate that Gr-1+CD11b+ myeloid cells are recruited into mammary carcinomas with type II TGF beta receptor gene (Tgfbr2) deletion and directly promote tumor metastasis. Gr-1+CD11b+ cells infiltrate into the invasive front of tumor tissues and facilitate tumor cell invasion and metastasis through a process involving metalloproteinase activity. This infiltration of Gr-1+CD11b+ cells also results in increased abundance of TGF beta 1 in tumors with Tgfbr2 deletion. The recruitment of Gr-1+CD11b+ cells into tumors with Tgfbr2 deletion involves two chemokine receptor axes, the SDF-1/CXCR4 and CXCL5/CXCR2 axes. Together, these data indicate that Gr-1+CD11b+ cells contribute to TGFbeta-mediated metastasis through enhancing tumor cell invasion and metastasis.

Figure 1. Gr-1+CD11b+ myeloid cells are recruited to mammary carcinomas with genetic deletion of Tgfbr2 (PyVmT/Tgfbr2^MGKO)

A: Flow cytometry analysis of infiltrating Gr-1+CD11b+ cells in PyVmT/Tgfbr2^MGKO tumors and PyVmT/Tgfbr2^flox/flox control tumors. Shown are representative flow cytometry plots. B: Quantitative data for the presence of Gr-1+CD11b+ cells in tumors as shown in 1A. Five to seven week old mice were analyzed. C: Flow cytometry analysis of tumor-residing Gr-1+CD11b+ cells in PyVmT/Tgfbr2^MGKO tumors. D: IHC of Gr-1+CD11b+ cells in the invasive front of PyVmT/Tgfbr2^MGKO mammary carcinomas compared with PyVmT/Tgfbr2^flox/flox tumors. Scale bars are indicated in the figures. E and F: Flow cytometry analysis of infiltrating F4/80 and VEGFR1 positive cells in PyVmT/Tgfbr2^MGKO tumors and PyVmT/Tgfbr2^flox/flox control tumors. Representative flow cytometry plots are shown on left and quantitative data on the right. m.g: adjacent mammary gland tissue; tu: tumor tissues. All quantitative data are presented with the mean ± standard error.

Figure 2. Gr-1+CD11b+ cells promote tumor metastasis

A: Increased Gr-1+CD11b+ cells in spleens and bone marrow of 4T1 tumor-bearing mice after tumor inoculation. Left panel: Flow cytometry analysis of Gr-1+CD11b+ cells in the spleen of tumor bearing mice 28 days following engraftment (4 or more mice per time point). Middle panel: spleen; Right panel: bone marrow (b.m.). B: Gr-1+CD11b+ cells in 4T1 tumors with flow cytometry analysis. C: IHC showing Gr-1+CD11b+ cells in the invasive front of 4T1 tumors 15 day after tumor inoculation. Scale bar, 100 μM. D: Single cell sorting of Gr-1+CD11b+ cells from tumors and spleens of 4T1 tumor-bearing mice 35 days after tumor inoculation. Flow cytometry analysis of Gr-1+CD11b+ cells after sorting is shown. E: Metastasis was significantly increased when 4T1 cells were co-injected with tumor derived Gr-1+CD11b+ cells including Gr-1+CD11b+ cells derived from tumor tissues (tu inf Gr-1+CD11b+ cells) spleens of tumor-bearing mice (tu sp Gr-1+CD11b+ cells). 4T1 cells alone and Gr-1+CD11b+ cells from normal mice (nor Gr-1+CD11b+ cells) were used as controls. Number of animals used is indicated in the bar. m.g: adjacent mammary gland tissue; tu: tumor tissues. Results are presented as the mean ± SE.

Figure 3. Gr-1+CD11b+ myeloid cells increased 4T1 tumor invasion in vivo and in vitro

A: Increased growth of recurrent tumors with 4T1 cells co-injected with Gr-1+CD11b+ cells derived from tumor tissues, or spleens of tumor-bearing mice. Number of animals is indicated in the bar. Results are presented as the mean ± SE. B: Fluorescent microscopy of 4T1 cells that invaded through a matrigel-coated transwell when co-cultured with tumor-derived Gr-1+CD11b+ cells (B-b) compared to normal Gr-1+CD11b+ cells (B-a). 4T1 cells were labeled with a green tracking dye, and Gr-1+CD11b+ cells with a red tracking dye. B-c: Close interaction between 4T1 cells and Gr-1+CD11b+ cells. Scale bar, 50 μM for all figures. C: An MMP inhibitor (GM6001, 1 mM) blocked Gr-1+CD11b+ cell promoted 4T1 cell invasion in vitro. Results are from two experiments with triplicates for each group, are presented as the mean ± SE. tu inf Gr-1+CD11b+ cells: Gr-1+CD11b+ cells from tumor tissues; tu sp Gr-1+CD11b+ cells: Gr-1+CD11b+ cells from spleens of tumor-bearing mice; nor Gr-1+CD11b+ cells: Gr-1+CD11b+ cells from spleens of normal mice. m.g: adjacent mammary gland tissue; tu: tumor tissues.

Figure 4. Increased production and function of MMPs in tumor residing Gr-1+CD11b+ cells

Real time RT-PCR of MMP14 and MMP2 (A) as well as MMP13 (B) in Gr-1+CD11b+ cells derived from tumor tissues compared with those from spleens. Results are presented as the mean ± SE. C. In situ zymography of MMPs in 4T1 mammary carcinomas and PyVmT/Tgfbr2^MGKO carcinomas. Abundant green fluorescence (indicator of MMP activities) was observed in the invasive front of 4T1 tumors (C-a & c) and PyVmT/Tgfbr2^MGKO carcinomas (C-d). Nuclei were labeled with 7AAD (red). Tumor sections treated with EDTA were used as negative controls (C-e). Scale bar, 50 uM for all figures. m.g: adjacent mammary gland tissue; tu: tumor tissues.

Figure 5. Increased TGFβ1 production in mammary carcinomas with genetic deletion of Tgfbr2

A: Increased TGFβ1 production in PyVmT/Tgfbr2^MGKO tumors compared with PyVmT/Tgfbr2^flox/flox tumors. n=4 mice per group. B left panel: Gr-1+CD11b+ cells from spleens of mice bearing large 4T1 tumors exhibited significantly higher TGFβ1 production when compared with Gr-1+CD11b+ cells from normal spleens. n=2 mice per group. B right panel: No difference of TGFβ1 production was found between tumor cell lines derived from PyVmT/Tgfbr2^MGKO tumors vs PyVmT/Tgfbr2^flox/flox tumors. TGFβ1 in conditioned media was measured by ELISA. Results are presented as the mean ± SE. C: Gr-1+CD11b+ cells are likely the resource for increased TGFβ13 production in mammary carcinomas with genetic deletion of Tgfbr2. IHC of TGFβ1 (left panel) showing TGFβ1 positive cells are mostly in the invasive front where Gr-1+CD11b+ cells were mostly present (right panel with Gr-1 staining). Scale bar, 50 uM for both figures. m.g: adjacent mammary gland tissue; tu: tumor tissues.

Figure 6. Mechanisms of recruitment of Gr-1+CD11b+ cells to the tumor microenvironment

A: Elevated production of CXCL5 in cell culture supernatant of PyVmT/Tgfbr2^MGKO mammary carcinomas. B: In vitro migration of Gr-1+CD11b+ cells in response to CXCL5. Gr-1+CD11b+ cells migration after 6–8 hr incubation were counted and plotted. Shown is one of the representative experiments of three performed. C: Inhibition of Gr-1+CD11b+ cell recruitment to PyVmT/Tgfbr2^MGKO tumors with CXCR2 antagonism. Percentage of Gr-1+CD11b+ cells in all cells from PyVmT/Tgfbr2^MGKO and control tumors were plotted. Mice bearing PyVmT/Tgfbr2^MGKO and control tumors were treated with a CXCR2 specific antagonist SB-265610 at 2 mg/kg/day for two weeks through I.P. D: Flow cytometry analysis of CXCR4 expression in infiltrating Gr-1+CD11b+ cells from 4T1 tumors as well as PyVmT/Tgfbr2^MGKO tumors, compared with those from spleens of the same tumor-bearing mice (as labeled). Histogram of CXCR4 expression was gated on Gr-1+CD11b+ double positive cells. Shown is one of the representative mice analyzed, n=3–5 mice per group. E: In vitro migration of Gr-1+CD11b+ cells in response to SDF-1. Gr-1+CD11b+ cells migration after 6–8 hr incubation were counted and plotted. Shown is one representative experiment of three performed. F: PyVmT/Tgfbr2^MGKO tumor metastasis with blockade of CXCR2 or CXCR4 alone or both. Tumor nodules in lung were counted after mice bearing 14-Day tumors were treated with CXCR2 or CXCR4 antagonists or both for 3 weeks. All results are presented as the mean ± SE.

Figure 7. Immature myeloid cells in human breast tumor tissues

A: IHC staining of myeloperoxidase to identify bone marrow-derived immature myeloid cells at the invasive front of human breast ductal adenocarcinomas (a & d). H&E staining of sequential tumor sections figure b to a, and e to d, respectively. Figure c and f are negative controls. Scale bar, 100 uM for all figures. B: Flow cytometry analysis of immature myeloid cells from single cell suspension of human breast ductal adenocarcinomas (stage 2–3). The analyzed cells were gated as 7AAD negative (top left panel, P1), negative for lineage markers including CD3 for T cell, CD19 for B cell, CD56 for NK cell, CD40/CD86/HLA-DR for dendritic cells, and CD14 for monocytes, and CD33, CD34 and CD15 positive (top right panel, P2). A histogram for FITC positive myeloid cells is shown in the lower right pane, with isotype control in the lower left panel.

Figure 8. Mechanisms for enhanced tumor progression resulting from loss of TGFβ signaling

Deletion of the type II TGFβ receptor gene in mammary carcinomas results in elevated production of CXCL5, which interact with CXCR2 on Gr-1+CD11b+ cells thus recruiting the cells into the tumor microenvironment. Gr-1+CD11b+ cells, as hemaetopoietic cells, express high level of CXCR4 that interact with SDF-1 in the tumor microenvironment, and are also recruited. Gr-1+CD11b+ cells promote tumor invasion through high expression of MMP14, MMP13 and MMP2. In addition, Gr-1+CD11b+ cells produce high levels of TGFβ1 that inhibit host immune surveillance.