TGFβ signaling in myeloid cells regulates mammary carcinoma cell invasion through fibroblast interactions

Abstract

Metastasis is the most devastating aspect of cancer, however we know very little about the mechanisms of local invasion, the earliest step of metastasis. During tumor growth CD11b+ Gr1+ cells, known also as MDSCs, have been shown to promote tumor progression by a wide spectrum of effects that suppress the anti-tumor immune response. In addition to immunosuppression, CD11b+ Gr1+ cells promote metastasis by mechanisms that are currently unknown. CD11b+ Gr1+ cells localize near fibroblasts, which remodel the ECM and leave tracks for collective cell migration of carcinoma cells. In this study we discovered that CD11b+ Gr1+ cells promote invasion of mammary carcinoma cells by increasing fibroblast migration. This effect was directed by secreted factors derived from CD11b+ Gr1+ cells. We have identified several CD11b+ Gr1+ cell secreted proteins that activate fibroblast migration, including CXCL11, CXCL15, FGF2, IGF-I, IL1Ra, Resistin, and Shh. The combination of CXCL11 and FGF2 had the strongest effect on fibroblast migration that is associated with Akt1 and ERK1/2 phosphorylation. Analysis of subsets of CD11b+ Gr1+ cells identified that CD11b+ Ly6Chigh Ly6Glow cells increase fibroblast migration more than other myeloid cell populations. Additionally, tumor-derived CD11b+ Gr1+ cells promote fibroblast migration more than splenic CD11b+ Gr1+ cells of tumor-bearing mice. While TGFβ signaling in fibroblasts does not regulate their migration toward CD11b+ Gr1+ cells, however deletion of TGFβ receptor II on CD11b+ Gr1+ cells downregulates CXCL11, Shh, IGF1 and FGF2 resulting in reduced fibroblast migration. These studies show that TGFβ signaling in CD11b+ Gr1+ cells promotes fibroblast directed carcinoma invasion and suggests that perivascular CD11b+ Ly6Chigh Ly6Glow cells may be the stimulus for localized invasion leading to metastasis.

Fig 1. CD11b⁺Gr1⁺ cells increase migration of fibroblasts and invasion of co-cultured carcinoma cells.

(A) CD11b⁺Gr1⁺ cells were isolated from spleen of tumor-bearing mice by magnetic separation and cultured for 16–18 hours. (B) Fibronectin-coated 8 uM pore Transwells were placed on top of cultured CD11b⁺Gr1⁺ cells and 5x10⁴ mammary fibroblasts were allowed to migrate for 5 hours. (C) CD11b⁺Gr1⁺ cells dose-dependently increase fibroblast migration. (D) Fibroblasts with deletion of TGFβRII have a further increase in migration in response to CD11b⁺Gr1⁺ cells. (E) Conditioned medium (CM) collected from CD11b⁺Gr1⁺ cells increases fibroblast migration to the same extent as live CD11b⁺Gr1⁺ cells. * – p<0.05, ** – p<0.01, *** – p<0.001 compared to no CD11b⁺Gr1⁺ cells. (F) Matrigel-coated 8 uM pore Transwells were placed on top of CD11b⁺Gr1⁺ CM and mammary carcinoma cells, fibroblasts or both cell types were allowed to invade for 18 hours. (G) CD11b⁺Gr1⁺ cells increase fibroblasts invasion. (H) CD11b⁺Gr1⁺ cells do not increase the invasion of carcinoma cells. (I) CD11b⁺Gr1⁺ cells increase carcinoma cell invasion when fibroblasts are co-cultured. * – p<0.05, ** – p<0.01, compared to open bars. (J) Western immunoblot of fibroblast Erk1/2 and Akt phosphorylation shows activation after 5 minutes of treatment with CM of CD11b⁺Gr1⁺ cells.

Fig 2. Cytokines and chemokines secreted by CD11b⁺Gr1⁺ cells increase fibroblast migration.

(A) Antibody array analysis of proteins secreted by CD11b⁺Gr1⁺ cells compared to fibroblasts. Although 144 proteins were assayed, only proteins predominantly expressed in CD11b⁺Gr1⁺ cells are shown. (B) Purified cytokine treatment of fibroblasts increases migration through Transwell chambers. (C) Combinations of several cytokines and chemokines increase fibroblast migration beyond treatment with a single cytokine/chemokine. Concentrations are same as on (B), * – p<0.05, ** – p<0.01, *** – p<0.001 compared to untreated; #: p<0.01 compared to all other treatments. (D) Inhibition of FGFR1 Kinase by PD173074 (5 nM) prevents fibroblast migration towards conditioned medium from CD11b⁺Gr1⁺ cells. ** – p<0.01, *** – p<0.001 relative to control medium.

Fig 3. TGFβRII signaling in Ly6C^highLy6G^low cells regulates the secretion of chemokines and results in increased fibroblast migration.

(A) FACS plot showing collection of Ly6C⁺, Ly6G⁺ and monocytes from spleen of 4T1 tumor-bearing mice. The plot was gated for CD45⁺ and CD11b⁺ cells. (B) FACS plot showing collection of T cells (CD3⁺) and B cells (CD19⁺) from spleen of 4T1 tumor-bearing mice. The plot was gated for CD45⁺ cells. (C) FACS plot showing collection of CD11b⁺Gr1⁺ cells and macrophages (CD11b⁺Gr1⁺) from 4T1 tumor tissue. The plot was gated for CD45⁺ cells. (D) Fibroblast migration to conditioned medium (from 1x10⁶ cells) prepared from subsets of splenocytes or myeloid cells from spleen and tumor tissue. Ly6C⁺ cells from spleen, tumor-derived CD11b⁺Gr1⁺ cells and tumor macrophages induce the most fibroblast migration. * – p<0.05 compared to control. (E) qRT-PCR analysis of cytokines/chemokines in splenic Ly6G (CD11b⁺Ly6G^highLy6C^low) and Ly6C (CD11b⁺Ly6C^highLy6G^low) cells. (F) qRT-PCR analysis of cytokines/chemokines in splenic CD11b⁺Gr1⁺ cells stimulated by TGFβ1 (1 ng/ml) for 18hr. with and without SB431542 (10 uM) * – p<0.05 compared with untreated cells, ** – p<0.05 compared with TGFβ treated cells, # – p<0.05 compared with WT cells. (G) CD11b⁺Gr1⁺ cells (4x10⁶) isolated from spleen of LLC tumor bearing mice were incubated 18hr in presence of TGFβ (1ng/ml) and TGFβ and SB431542 (10uM). Level of FGFb was measured by ELISA (R&D System, Minneapolis, MN). (H) CD11b⁺Gr1⁺ cells (3x10⁶) isolated from spleen of LLC tumor bearing mice with deleted TGFβRII decrease migration of fibroblasts compared with CD11b⁺Gr1⁺ cells with intact TGFβ signaling. “-” control—negative control, DMEM without serum, * – p<0.01. TGFβ1 (1 ng/ml), SB431542 (10 uM).