Fatty acid-binding protein E-FABP restricts tumor growth by promoting IFN-β responses in tumor-associated macrophages

Abstract

Fatty acid-binding proteins (FABP) are known central regulators of both metabolic and inflammatory pathways, but their role in tumor development remains largely unexplored. Here, we report that host expression of epidermal FABP (E-FABP) protects against mammary tumor growth. We find that E-FABP is highly expressed in macrophages, particularly in a specific subset, promoting their antitumor activity. In the tumor stroma, E-FABP-expressing tumor-associated macrophages (TAM) produce high levels of IFN-β through upregulation of lipid droplet formation in response to tumors. E-FABP-mediated IFN-β signaling can further enhance recruitment of tumoricidal effector cells, in particular natural killer cells, to the tumor stroma for antitumor activity. These findings identify E-FABP as a new protective factor to strengthen IFN-β responses against tumor growth.

Figure 1. E-FABP protects mice from mammary tumor growth

A, schematic of the experimental procedure. B, E0771 cells (0.2×10⁶) were orthotopically injected into the mammary pad of E-FABP^−/− and WT mice (n=9/group). Tumor growth was measured at 3 day intervals. Weight of tumor mass (C), tumor metastasis spots (D) and H&E staining of lungs (E) were analyzed on day 24 after E0771 cell implantation in mice. Representative lung images (F) and tumor spots in lungs (G) on day 18 after tail vein injection of E0771 cells (0.2×10⁶) in mice. Data represent mean ± SD (*, p < 0.05)

Figure 2. Analysis of E-FABP expression profile

A, real-time PCR analysis of E-FABP expression in splenic immune cells separated by a flow sorter. Adipocytes isolated from normal mammary fat pad were used as a control. B, analysis of F4/80⁺ macrophages (green) and E-FABP expression (red) in mammary fat tissues by confocal microscopy. C, analysis of E-FABP expression (red) in immune cells (nuclei,DAPI) separated from peripheral blood of mice. Analysis of E-FABP expression in bone marrow-derived macrophages by intracellular staining (D) and confocal microscopy (E). Phenotype analysis of immune cells in the spleen (F) and draining lymph nodes (dLN) (G) of naïve and tumor-bearing mice (3 weeks post E0771 cell implantation) by flow cytometry (n=6/group). Analysis of F4/80⁺ macrophages by immunohistochemistry staining (H) and immune cell populations by flow cytometry (I) in tumors 3 weeks after E0771 cell implantation. Data represent mean ± SD (*, p < 0.05).

Figure 3. E-FABP is highly expressed in specific subsets of macrophages

A, identification of CD11b⁺F4/80⁺ macrophages from mouse spleen. B, distinct subsets of splenic macrophages by Ly6C and MHC-II staining. Analyses of CD11c expression by flow histogram (C) and mean fluorescent intensity (MFI) (D), and E-FABP expression by real-time PCR (E) in distinct macrophage subsets. F, flow cytometric analysis of dynamic changes of distinct subsets of TAMs in tumor stroma at indicated time points after E0771 cell implantation. Analysis of CD11c expression by MFI (G) and E-FABP expression by real-time PCR (H) in distinct subsets of TAMs on day 7 post E0771 cell implantation.

Figure 4. Microarray analysis of E-FABP-regulated genes in macrophages

A, a heat map of differentiated genes in E0771-stimulated GM-BMMs by Affymetrix microarray analysis. B, real-time PCR confirmation of interested E-FABP-regulated genes identified by microarray. C, IFNβ levels in the supernatants collected from GM-BMMs after stimulation with E0771 lysates for 3 hours by ELISA. D, analysis of E-FABP expression in macrophages transfected with E-FABP siRNA or scrambled oligos by western blotting. E, IFNβ levels in supernatants collected from macrophages as described in panel D in response to LPS at indicated time points. Data represent mean ± SD (*, p < 0.05, **, p < 0.01).

Figure 5. E-FABP promotes IFNβ responses by enhancing LD formation in macrophages

GM-BMMs were cultured with or without saturated FAs (stearic acid, 100µM) or unsaturated FAs (oleic/linoleic acid, 25µM) for 24 hr. E-FABP expression (A), LD formation (B), Viperin (C) and IFNβ (D) expression in these cells were measured by real-time PCR or confocal microscopy. E, analysis of IFNβ levels in supernatants of unsaturated FA-treated macrophages in the absence or presence of indicated LD inhibitor (Triacsin C) by ELISA. Analysis of LD formation (F) and Viperin expression (G) in GM-BMMs after coculture with or without E0771 tumor cells in a transwell for 18 hours by confocal microscopy. Analysis of phosphorylation of STAT1 (H), STAT2 (I), and their total proteins in GM-BMMs after stimulation with 100U IFNβ for the indicated time periods. Expression of E-FABP (J), Viperin (K), IRF7 (L), IFNβ (M), CXCL10 (N), CXCL11 (O) in the Q2 subset of TAMs (separated from tumors on day 7 post E0771 cell implantation) was analyzed by real-time PCR. Data represent mean ± SD (*, p < 0.05, **, p < 0.01).

Figure 6. E-FABP enhances recruitment of tumoricidal effector cells

Analysis of infiltrated CD4⁺ T cell (A), CD8⁺ T cells (B) and NK cells (C) in tumors on day 7 post E0771 cell implantation in mice by flow staining. The percentage of each population was shown in the right panel. D, target cells (E0771) were labeled with CFSE and cocultured with effector cells (collected from dLNs of E0771-tumor bearing mice) at the indicated ratio. The percentage of specific tumor killing was analyzed by a flow cytometer. E, tumor specific killing assays as described in panel D (effect/target ratio:100) were performed in the absence or presence of respective blocking antibody to CD4, CD8, NK1.1 and TRIAL. F, Percentage of NK cells in spleen, peripheral blood and lymph nodes in mice with or without NK cell depletion. G, measurement of tumor size two weeks after E0771 cell implantation in WT and E-FABP^−/− mice with or without NK cell depletion.

Figure 7. E-FABP expression in TAMs of human breast cancer

A, analysis of E-FABP expression in normal breast tissues and various types of malignant breast tissues from the publicly accessible microarray databases (www.oncomine.org/resouce). B, E-FABP expression in the stroma of normal and malignant breast tissues by analyzing GEO dataset GSE9014. C, co-staining of E-FABP expression (red) and TAMs (green) in different stages of invasive breast cancer tissues by confocal microscopy. H&E staining of the same section was shown on the left panel. D, numbers of E-FABP⁺ TAMs per high power fields (×400 magnification) were shown as mean ± SD. *p<0.05 as compared to stage III invasive breast cancers.