Cancer associated fibroblasts promote tumor growth and metastasis by modulating the tumor immune microenvironment in a 4T1 murine breast cancer model

Abstract

Background: Local inflammation associated with solid tumors commonly results from factors released by tumor cells and the tumor stroma, and promotes tumor progression. Cancer associated fibroblasts comprise a majority of the cells found in tumor stroma and are appealing targets for cancer therapy. Here, our aim was to determine the efficacy of targeting cancer associated fibroblasts for the treatment of metastatic breast cancer.

Methodology/principal findings: We demonstrate that cancer associated fibroblasts are key modulators of immune polarization in the tumor microenvironment of a 4T1 murine model of metastatic breast cancer. Elimination of cancer associated fibroblasts in vivo by a DNA vaccine targeted to fibroblast activation protein results in a shift of the immune microenvironment from a Th2 to Th1 polarization. This shift is characterized by increased protein expression of IL-2 and IL-7, suppressed recruitment of tumor-associated macrophages, myeloid derived suppressor cells, T regulatory cells, and decreased tumor angiogenesis and lymphangiogenesis. Additionally, the vaccine improved anti-metastatic effects of doxorubicin chemotherapy and enhanced suppression of IL-6 and IL-4 protein expression while increasing recruitment of dendritic cells and CD8(+) T cells. Treatment with the combination therapy also reduced tumor-associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression.

Conclusions/significance: Our findings demonstrate that cancer associated fibroblasts promote tumor growth and metastasis through their role as key modulators of immune polarization in the tumor microenvironment and are valid targets for therapy of metastatic breast cancer.

Figure 1. Vaccination with pFap enhances the anti-metastaic effects of doxorubicin chemotherapy.

(A) Mice were treated in a prophylactic setting and sacrificed 25 days after tumor cell challenge. Primary tumors were resected and their weight (TW) was compared to body weight (BW) to calculate tumor burden. (B) Mice were treated in a therapeutic setting where primary tumors were allowed to establish prior to resection and treatment with the combination therapy. Survival of mice after tumor resection was measured using Kaplan-Meier survival curves. *, p<0.05. (C) Lungs were isolated from moribund mice treated in a therapeutic setting. To visualize metastatic foci, lungs were sectioned and stained with hematoxylin and eosin. Scale bar = 1 mm. (D) Surface areas (SA) of metastatic foci and lung were measured using ImageJ software (n = 5 mice/group). Results are depicted as percent SA^metastasis/SA^lung. ***, p<0.0005.

Figure 2. Vaccination with pFap results in Th2 to Th1 cytokine transition in the tumor microenvironment.

Mice were treated in a prophylactic setting and primary tumors isolated 25 days later. (A–B) Tumor homogenates were analyzed by Western blotting to determine IL-6 and IL4 (A) and IL-2 and IL-7 (B) protein levels. (C) Live primary tumor cell suspensions were analyzed by flow cytometry using anti-CD8 and anti-CD25 antibodies to detect activated T-cells. Results are shown as percent of CD8⁺/CD25⁺ T cells relative to mice treated with the combination therapy. (D) Splenocytes were isolated from tumor bearing mice treated with the combination therapy or controls. Splenocytes were stimulated twice with IL-2 and then incubated with irradiated 4T1 tumor cells for 24 hours prior to detection of CD8⁺/Granzyme B⁺ cells by flow cytometry. (E) Primary tumors were isolated from mice treated in a prophylactic setting 10-days after the final treatment with doxorubicin and apoptotic tumor cells were visualized by quantified using the TUNEL assay. (F) Additionally, we also used immunohistochemistry to visualize CD8⁺ T cells (red) and apoptotic tumor cells expressing caspase 3 (green) in primary tumors. Scale bar = 100 µm.

Figure 3. Vaccination with pFap suppresses TAM, MDSC, Treg, and enhances DC and CTL recruitment.

Primary tumors were isolated from mice 25 days after orthotopic challenge and immune cells were identified in primary tumors using antibodies for the following: (A) tumor-associated macrophages (TAMs): F4/80 (red), (B) myeloid derived suppressor cells (MDSCs): CD11b (red, nuclear) and Gr-1 (green, nuclear), (C) T regulatory cells (Tregs, white arrowheads): CD4 (red, cell surface) and FOXP3 (green, nuclear), (D) dendritic cells (DC): 33D1 (red), and (E) cytotoxic T lymphocytes (CTL): CD8 (red) Scale bar for all panels = 100 µm.

Figure 4. Combination therapy reduces tumor associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression.

Tumors were isolated 25 days after orthothopic challenge and total RNA was isolated from the stroma (S) and tumor cells (TC) by laser capture dissection microscopy, and used to generate cDNA for qRT-PCR analysis. Gene expression is normalized to actin and shown relative to stroma of vector control for Vegfa, Pdgfc, and GM-CSF (A–C, left panel, respectively). *, p<0.05, **, p<0.005. Whole cell extracts were derived from primary tumors and subjected to immunoblotting to detect Vegf, Pdgfc and GM-CSF protein and detection of actin was used as a loading control (A–C, right panel, respectively).

Figure 5. Vaccination with pFap suppresses tumor angiogenesis and lymphangiogenesis.

Mice were treated with vector control (A), pFap (B), doxorubicin (C), or pFap plus doxorubicin (D). Primary tumors were isolated from mice 25 days after orthotopic challenge. Tumor sections were treated with anti-CD31 and anti-LYVE1 antibodies to detect blood (brown) and lymph (green) vessels, respectively. Scale bar = 100 µm.

Figure 6. Our combination therapy exerts its anti-metastatic effects through modulation of the immune tumor microenvironment.

Cancer associated fibroblasts (CAFs) promote tumor growth and metastasis by recruiting tumor associated macrophages (TAMS), myeloid derived suppressor cells (MDSCs) and T regulatory cells (Tregs) and promoting Th2 polarization of the tumor microenvironment. This modulation of the tumor microenvironment results in production of growth factors and cytokines that support tumor growth and metastasis by promoting angiogenesis, lymphangiogenesis, and suppression of anti-tumor immune responses. Elimination of CAFs by treatment with our pFap vaccine, in combination with doxorubicin chemotherapy, modulates the immune tumor microenvironment and shifts polarization from Th2 to Th1, characterized by an increase in dendritic cells (DCs) and cytotoxic T lymphocytes (CTLs) and Th1 cytokine expression. As a result, tumor angiogenesis and lymphangiogenesis are reduced and anti-tumor immune responses are enhanced leading to suppression of spontaneous metastasis of 4T1 breast cancer cells.