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. 2021 Jun;9(6):e002548.
doi: 10.1136/jitc-2021-002548.

S100A4 enhances protumor macrophage polarization by control of PPAR-γ-dependent induction of fatty acid oxidation

Shuangqing Liu #  1   2   3 , Huilei Zhang #  1   2 , Yanan Li  1 , Yana Zhang  3 , Yangyang Bian  3 , Yanqiong Zeng  4 , Xiaohan Yao  3 , Jiajia Wan  3 , Xu Chen  1   2 , Jianru Li  1   2 , Zhaoqing Wang  5   2 , Zhihai Qin  5   2   3   4
Affiliations

S100A4 enhances protumor macrophage polarization by control of PPAR-γ-dependent induction of fatty acid oxidation

Shuangqing Liu et al. J Immunother Cancer. 2021 Jun.

Abstract

Background: The peroxisome proliferator-activated receptor γ (PPAR-γ)-dependent upregulation of fatty acid oxidation (FAO) mediates protumor (also known as M2-like) polarization of tumor-associated macrophages (TAMs). However, upstream factors determining PPAR-γ upregulation in TAM protumor polarization are not fully identified. S100A4 plays crucial roles in promotion of cancer malignancy and mitochondrial metabolism. The fact that macrophage-derived S100A4 is major source of extracellular S100A4 suggests that macrophages contain a high abundance of intracellular S100A4. However, whether intracellular S100A4 in macrophages also contributes to cancer malignancy by enabling TAMs to acquire M2-like protumor activity remains unknown.

Methods: Growth of tumor cells was evaluated in murine tumor models. TAMs were isolated from the tumor grafts in whole-body S100A4-knockout (KO), macrophage-specific S100A4-KO and transgenic S100A4WT-EGFP mice (expressing enhanced green fluorescent protein (EGFP) under the control of the S100A4 promoter). In vitro induction of macrophage M2 polarization was conducted by interleukin 4 (IL-4) stimulation. RNA-sequencing, real-time quantitative PCR, flow cytometry, western blotting, immunofluorescence staining and mass spectrometry were used to determine macrophage phenotype. Exogenous and endogenous FAO, FA uptake and measurement of lipid content were used to analyze macrophage metabolism.

Results: TAMs contain two subsets based on whether they express S100A4 or not and that S100A4+ subsets display protumor phenotypes. S100A4 can be induced by IL-4, an M2 activator of macrophage polarization. Mechanistically, S100A4 controls the upregulation of PPAR-γ, a transcription factor required for FAO induction during TAM protumor polarization. In S100A4+ TAMs, PPAR-γ mainly upregulates CD36, a FA transporter, to enhance FA absorption as well as FAO. In contrast, S100A4-deficient TAMs exhibited decreased protumor activity because of failure in PPAR-γ upregulation-dependent FAO induction.

Conclusions: We find that macrophagic S100A4 enhances protumor macrophage polarization as a determinant of PPAR-γ-dependent FAO induction. Accordingly, our findings provide an insight into the general mechanisms of TAM polarization toward protumor phenotypes. Therefore, our results strongly suggest that targeting macrophagic S100A4 may be a potential strategy to prevent TAMs from re-differentiation toward a protumor phenotype.

Keywords: immunity; immunomodulation; innate; macrophages; metabolic networks and pathways; tumor microenvironment.

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Conflict of interest statement

Competing interests: No, there are no competing interests.

Figures

Figure 1
Figure 1
S100A4+ TAMs possess protumor activity. (A) Flow cytometric analysis of the frequency of immune cell populations in tumor-infiltrating S100A4+ cells of four mice. (B) Flow cytometric analysis of TAMs frequency in S100A4WT-EGFP cells by double staining with CD11b and F4/80. (C) Flow cytometric analysis of the frequency of S100A4+ TAMs (CD11b+/F4/80+) and the fluorescence intensity of CD206 in S100A4+ and S100A4− TAMs. (D–F) Growth of tumor grafts was monitored over time after the initial injection of E0771 breast cancer cells into S100A4WT or S100A4KO female mice (n≥5). tumor weight and representative pictures of tumor grafts excised at the end of the experiment. (G) Growth of tumor grafts was monitored over time after initial inoculation of E0771 breast cancer cells into S100A4M-WT or S100A4M-KO female mice (n≥8). Data are presented as mean±SE and were analyzed by unpaired non-parametric Mann-Whitney U test in C, E or two-way ANOVA Sidak's multiple comparisons in D, G. The data are from one representative experiment of more than three independent experiments (B–G). *P<0.05, **P<0.01, ***P<0.001. SSC, side scatter; ANOVA, analysis of variance; GMFI, geometric mean fluorescence intensity; KO, knockout; TAM, tumor-associated macrophage; WT, wild type.
Figure 2
Figure 2
Macrophagic deficiency of S100A4 impairs TAM alternative activation. (A–D) S100A4WT or S100A4KO Raw264.7 cells were activated with IL-4 (20 ng/mL). Nonactivated macrophages were used as controls. A Lack of S100A4 expression was analyzed by immunofluorescence in S100A4WT or S100A4KO Raw264.7 cells (middle). The immune complexes were detected with a secondary antibody conjugated to Alexa Fluor 555 (red). DNA was stained with DAPI (blue). Scale bars, 10 μm. Expression of S100A4 mRNA and protein was analyzed by q-PCR (left in B) and immunoblotting (right in B), respectively. Band densities (mean±SE) for S100A4 were measured in at least three independent immunoblots and normalized to those of β-actin (loading control). Arginase activity was assessed with or without 36-hours activation with IL-4 in (C). Expression of M2 hallmarks was assessed by q-PCR after 36-hours activation with IL-4 in D. The values were normalized with that in S100A4WT Raw264.7 cells with IL-4 stimulation. (E–H) S100A4WT or S100A4KO BMDMs were treated with IL-4 (20 ng/mL, E) or IFN-γ (20 ng/mL) in combination with LPS (100 ng/mL, F). S100A4 mRNA expression was analyzed by q-PCR in E, F. The values were normalized with that in S100A4WT BMDMs without stimulation. After 36-hours activation with IL-4, the expression of CD206 was examined and quantified by flow cytometry in G and expression of M2 hallmarks was assessed by q-PCR in H. (I, J) Flow cytometric analysis of CD206 expression in TAMs and the proportion of CD206+ TAMs (I) in the tumor grafts from E0771 breast cancer cell-bearing S100A4WT and S100A4KO female mice (n=5) or (J) S100A4M-WT and S100A4M-KO female mice (n=6). Data are presented as mean±SE and were analyzed by unpaired Student’s t-test in B–G, and H and by unpaired nonparametric Mann-Whitney U test in I, J. The data are from one representative experiment of more than three independent experiments (A–C, I, J). *P<0.05, **P<0.01, ***P<0.001, BMDMs, bone marrow-derived macrophages; GMFI, geometric mean fluorescence intensity; IFN-γ, interferon-γ; IL-4, interleukin 4; KO, knockout; n.s., not significant; TAM, tumor-associated macrophage; WT, wild type; DAPI, 4',6-diamidino-2-phenylindole.
Figure 3
Figure 3
Macrophagic S100A4 correlates with chemotherapy-resistance and poor prognosis. (A) Effects of deleting proliferating S100A4+ cells on chemosensitivity. Growth of tumor grafts was monitored over time after initial injection of TSA breast cancer cells into S100A4TK+ transgenic mice and control S100A4TK− littermates (n≥6). The mice were treated with doxorubicin (5 mg/kg) on days 11 and 13 before treatment with GCV (50 mg/kg body weight) on the indicated days. (B, C) Coinjection of 4T1 breast cancer cells together with S100A4WT or S100A4KO Raw264.7 cells (1:1 ratio) into WT Balb/c mice (n≥5) treated with doxorubicin (5 mg/kg) on days 8 and 11. Growth of tumor grafts was monitored over time after initial injection (B). Tumor weight of tumor grafts excised at the end of the experiment are shown (C). (D–F) Comparison of the relapse-free survival probability or overall survival rate of S100A4low/CD68low and S100A4high/CD68high patients with breast (D), ovarian (E), and lung (F) cancers after chemotherapy. Data are derived from a Kaplan-Meier plotter. Data (in A–C) are presented as mean±SE and were analyzed by unpaired Student’s t-test and two-way ANOVA Sidak's multiple comparisons. The data are from one representative experiment of three independent experiments (A–C). *P<0.05, **P<0.01, ***P<0.001. ANOVA, analysis of variance; GCV, ganciclovir; WT, wild type.
Figure 4
Figure 4
S100A4 depletion reduces macrophagic capability in usage of exogenous fatty acids. (A–D) RNA sequencing analysis of DEGs between S100A4+ and S100A4− TAMs in tumor grafts from E0771 breast cancer cell-bearing S100A4WT-EGFP reporter mice. Volcano diagram of DEGs, threshold is padj < 0.05 (A). Heatmap view of gene expression of M2 markers (B), oxidative phosphorylation (C) and FA metabolism (D) in S100A4+ and S100A4− TAMs. (E, F) S100A4WT or S100A4KO Raw264.7 cells were activated by IL-4 for 36 hours. The non-activated macrophages were used as controls. The mitochondrial oxygen consumption rate (OCR) was monitored and analyzed in the presence or absence of FAO substrate (PALM, palmitate) or CPT1 inhibitor (ETO, etomoxir) via XFe96 Analyzer (E). The basal and the maximal endogenous or exogenous FAO was quantified based on the OCR value (F). (G) Measurement of FA uptake in S100A4WT or S100A4KO Raw264.7 cells. (H, I) Flow cytometric analysis of lipid content in S100A4WT or S100A4KO Raw264.7 cells (H) or in BMDMs (I) stained with Nile red. (J) Identification of the re-expression of S100A4 in S100A4KO Raw264.7 cells. (K) Flow cytometric analysis of lipid content (Nile red staining) in S100A4WT, S100A4KO, or S100A4RE Raw264.7 cells with oleate (0.2 mM) incubation. (L) Flow cytometric analysis of lipid content in S100A4+ or S100A4− TAMs isolated from E0771 breast cancer cell-bearing female mice. Data are presented as mean±SE and were analyzed by two-way ANOVA with Tukey's multiple multiple comparisons and unpaired non-parametric Mann-Whitney U test. The data are from one representative experiment of three independent experiments (E–K). *P<0.05, ***P<0.001. ANOVA, analysis of variance; BMDMs, bone marrow-derived macrophages; DEGs, differentially expressed genes; FA, fatty acid; FAO, fatty acid oxidation; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; GMFI, geometric mean fluorescence intensity; IL-4, interleukin 4; KO, knockout; Oligo, oligomycin; n.s., not signifacant; Rtn/AA, rotenone/antimycin-A; TAM, tumor-associated macrophage; TGF-β, transforming growth factor-β; WT, wild type.
Figure 5
Figure 5
S100A4 controls macrophage alternative activation via determining PPAR-γ induction. (A) Immunoblotting examination of expression of PPARs and their co-activators in S100A4WT or S100A4KO Raw264.7 cells treated with or without IL-4. (B) Immunofluorescence analysis of expression of S100A4 and PPAR-γ in IL-4-activated S100A4WT or S100A4KO Raw264.7 cells. The immune complexes were detected with a secondary antibody conjugated to Alexa Fluor 555 (red). DNA was stained with DAPI (blue). Scale bars, 5 μm. (C) q-PCR analysis of PPAR-γ expression in IL-4-activated S100A4WT or S100A4KO BMDMs. (D) Flow cytometric analysis of PPAR-γ expression in TAMs of the tumor grafts isolated from E0771 breast cancer cell-bearing S100A4M-WT or S100A4M-KO mice. (E) Immunoblotting examination of PPAR-γ expression in IL-4-activated S100A4WT, S100A4KO or S100A4RE Raw264.7 cells. (F) q-PCR analysis of M2 marker expression in IL-4-activated BMDMs in the presence or absence of PPAR-γ inhibitor (mifobate, 200 µM; or T0070907, 100 µM) or agonist (troglitazone, 2 µM). Data are presented as mean±SE and were analyzed by one-way ANOVA with Tukey's multiple comparisons. The data are from one representative experiment of three independent experiments (A, B, E). *P<0.05, **P<0.01, ***P<0.001. ANOVA, analysis of variance; BMDMs, bone marrow-derived macrophages; GMFI, geometric mean fluorescence intensity; KO, knockout; IL-4, interleukin 4; MFI, mean fluorescence intensity; PPAR-γ, peroxisome proliferator-activated receptor γ; TAM, tumor-associated macrophage; WT, wild type; DAPI, 4',6-diamidino-2-phenylindole.
Figure 6
Figure 6
S100A4 promotes fatty acid uptake through upregulation of PPAR-γ targeting gene CD36. (A) Immunoblotting examination of CD36 expression in IL-4−activated S100A4WT or S100A4KO BMDMs. (B) Flow cytometric analysis of CD36 expression in S100A4+ and S100A4− TAMs from tumor grafts in E0771 breast cancer cell-bearing S100A4WT−EGFP reporter mice. (C) Flow cytometric analysis of CD36 expression in S100A4M−WT and S100A4 M−KO TAMs from tumor grafts in E0771 breast cancer cell-bearing S100A4M−WT or S100A4M−KO female mice (n≥5). (D) Immunoblotting examination of CD36 expression in IL-4-activated Raw264.7 cells. (E, F) Examination of CD36 mRNA and protein expression in IL-4-activated S100A4WT or S100A4KO BMDMs in the presence or absence of PPAR-γ inhibitor (mifobate, 200 µM; or T0070907, 100 µM) or agonist (troglitazone, 2 µM). (G) Flow cytometric analysis of lipid content in S100A4WT or S100A4KO BMDMs stained with Nile red. The cells were incubated with oleate (0.2 mM) in the presence or absence of CD36 inhibitor SSO (25 µM) as indicated. Data are from three independent experiments. The values were normalized with that in S100A4WT BMDMs without treatment. (H, I) E0771 breast cancer cells were implanted into S100A4M−WT (with or without PPAR-γ inhibitor, T0070907) or S100A4M−KO female mice. The growth of tumor grafts (H, left) was monitored over time after initial cell implantation, and the tumor weight (I, right) of tumor grafts excised at the end of the experiment are shown. The number of CD206+ protumor TAMs (J) in tumor grafts was examined by flow cytometry. Data are presented as mean±SE and were analyzed by unpaired nonparametric Mann-Whitney U test in B, C; one-way ANOVA with Tukey's multiple comparisons test in E, G–J; and two-way ANOVA with Dunnett's multiple comparisons test in H. The data are from one representative experiment of three independent experiments (A–D, F, J). *P<0.05, **P<0.01, ***P<0.001. ANOVA, analysis of variance; BMDMs, bone marrow-derived macrophages; GMFI, geometric mean fluorescence intensity; KO, knockout; IL-4, interleukin 4; MFI, mean fluorescence intensity; PPAR-γ, peroxisome proliferator-activated receptor γ; TAM, tumor-associated macrophage; WT, wild type; SSO, sulfo-N-succinimidyl oleate Na.
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