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

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.


Measurement of exogenous and endogenous fatty acid oxidation (FAO)
The respiration changes, caused by utilization of exogenous FAs, endogenous FAs, or uncoupling by FAs, were simultaneously measured using kit that works with the Seahorse XF analyzer and can simultaneously measure oxidation of exogenous and endogenous fatty acids (Agilent Technologies). The tested S100A4 WT and S100A4 KO Raw264.7 cells (10,000/well) were cultured on the XF 96 cell culture microplate (102601-100, Agilent, Palo Alto, California, USA) and were stimulated with or without IL-4 (20 ng/mL) for 36 hours. Then the cell culture medium was changed to substrate-limited medium and cells were further cultured in this medium for 7 hours.

Real-time quantitative PCR (q-PCR)
Total RNA was harvested from mouse tissues with Trizol reagent (Invitrogen). First-strand cDNA synthesis was performed using One-step RT-PCR kit (A215-01, GenStar, Beijing, China) ). Then cDNA was used as a template for q-PCR reactions using Taqman primer-probes against specified mRNA transcripts (Applied Biosystems, Waltham, Massachusetts, USA).
Reactions were performed using 2 × Real Star Green Power Mixture (A311-10, GenStar, Beijing, China). FAM channel intensity was normalized to ROX intensity, and Ct values were calculated using automatically determined threshold values using SDS software (Applied Biosystems). The relative expression of genes was calculated by a standard curve method and normalized to the expression level of GAPDH. Gene-specific PCR primers are listed in online supplemental Table 1.

Protein extraction and western blotting
For western blotting, cells were placed on iced and washed twice with chilled PBS.
Proteins were extracted with RIPA buffer (Cell Signaling Technology) plus protease inhibitor cocktail and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The protein concentration was determined with the BCA protein assay kit (Thermo Fisher Scientific). An estimated 15-50 μg proteins were loaded per well on SDS-PAGE gel and transferred onto PVDF membrane (Thermo Fisher Scientific). Membranes were washed with 1 × TBST, blocked with 5% BSA in TBST at room temperature for 1 hour, and incubated in first antibody as indicated in each figure and its legend. Next day, the membranes were washed 3 times with 1 × TBST, incubated with secondary antibody at 1:5000 dilution in 1 × TBST for 2 hours, and washed 3 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)

Immunofluorescence staining and immunohistochemistry
Cells were washed and fixed with 4% paraformaldehyde in PBS for 15 minutes, permeabilized with PBS/0.1% Triton X-100 for 10 minutes, and blocked with 3% BSA in PBS for 1 hour. The permeabilized cells were then incubated overnight at 4°C with the primary antibody against S100A4 (1:200) or PPAR-γ (1:200) in PBS containing 3% BSA. Cells were washed three times with PBS/0.1% Tween 20, and then incubated for 1 hour with a fluorescence-conjugated secondary antibody at a dilution of 1:300 at 37°C. After 45 minutes, the cells were washed with PBS/0.1% Tween 20, and counterstained with DAPI to detect DNA.
For immunohistochemistry, the murine tissues were fixed in 4% paraformaldehyde (pH 7.4) overnight, embedded in paraffin, and serially sectioned at 5 μm. For immunofluorescent staining, the sections were then incubated with primary antibodies against CD206 (1:200) and CD31 (1:200) in PBS with 3% BSA overnight at 4°C. The tissue sections were washed three times with PBS and then incubated for 1 hour with fluorescence-conjugated Alexa Fluor 555 goat anti-rat or Alexa Fluor 555 donkey anti-rabbit secondary antibody at a dilution of 1:300 at 37°C. Images of the stained tissue samples were obtained using an OLYMPUS DP71 microscope (Tokyo, Japan). The fluorescence intensity was calculated by the Image J software.

Macrophage preparation and activation
BMDMs were prepared from BM cells, isolated from mouse femur. Then the BM cells were cultured in 10 cm dishes with DMEM containing 20% FBS and M-CSF (50 ng/mL) medium for 6-7 days. The adherent macrophages were harvested and cultured for further experiments. Positive selection of macrophage was conducted by the flow-cytometric analysis.
For the activation experiments, macrophages were stimulated with IL-4 (20 ng/mL) for 36 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) hours, or with LPS (100 ng/mL) in combination with IFN-γ (20 ng/mL) for 12 hours.

Arginase activity assay
Relative arginine levels were measured using a colorimetric arginase activity assay kit according to manufacturer's instructions (ab180877, Abcam). The isolated cells were washed with PBS and were resuspended in assay buffer with 0.01% of Triton X-100 to disrupt the cell membrane. The supernatant samples were diluted 1:10 and 40 μL incubated with the arginase enzyme. After 10 minutes, the absorbance was measured at 570 nm in kinetic mode in an Epoch 2 microplate reader (BioTek, Winooski, Vermont, USA). The values were compared with an arginine standard curve.

Detection of cell proliferation with cell counting kit-8 (CCK-8) assay
Cells were incubated in serum-free medium for 24 hours and cells in the logarithmic growth stage were obtained and centrifuged for 5 minutes at 64 × g at room temperature. The cell density was adjusted to 2×10 4 cells/mL, and the suspensions were seeded into 96-well plates at 100 µL/well. After overnight, the cells were treated with doxorubicin (Dox) or 5fluorouracil (5Fu) for 24 hours, the untreated cells used as negative control. Then, 10 µL of CCK-8 detection solution (ab228554, Abcam) was added to each well and incubated at 37°C for 1.5 hours. The optical density (OD) value was read at 450 nm, and the cell survival rate was calculated as follows: (OD450 treated group/OD450 control group) × 100%.

Mass spectrometry (MS)
S100A4 WT and S100A4 KO BMDMs were stimulated with IL-4 (20 ng/mL). After 36 hours, the cells were washed with cold PBS twice and harvested by ice-cold lysis buffer (pH 8.5, 8 M urea in 100 mM Tris-HCl with protease inhibitor cocktail). The cell lysates were centrifuged at 15 000 g for 30 minutes at 4°C to remove cell debris. Supernatants were collected and sent to process with MS analysis by Applied Protein Technology (Shanghai, China). The filters were set as follows: peptides with cut-off of FDR = 0.01 and significance threshold P < 0.05 (with 95% confidence). and B220 (lower). The tumour grafts are isolated from E0771 breast cancer cell-bearing S100A4 WT-EGFP reporter mice. (B) Flow cytometric analysis of the frequencies of macrophages (stained with F4/80 and CD11b, right) in tumour-infiltrating S100A4 + cells isolated from MCA205 fibrosarcoma cell-bearing S100A4 WT-EGFP reporter mice. (C) Body weight of E0771 breast cancer cell-bearing S100A4 KO or S100A4 WT mice (n≥5) was measured every 2 days after 7-days inoculation. (D) Growth of tumour grafts in tumour-bearing S100A4 WT or S100A4 KO Scheme of the construction of S100A4 floxp mouse. Targeted mice were crossed with WT mice to generate S100A4-floxed mice, which were further crossed with Lysosome M (LysM) Cre mice to generate myeloid cells conditional knockout mice. (B) Genotyping PCR analysis of S100A4 f/f LysM-cre − and S100A4 f/f LysM-cre + mice. (C) Immunoblotting analysis of S100A4 protein expression in macrophages and lymphocytes isolated from S100A4 M-WT and S100A4 M-KO mice. The data are from one representative experiment of three independent experiments (B-C). protumour marker CD206 were detected by q-PCR in four S100A4 knockout monoclonal cell lines stimulated with IL-4 for 36 hours. (E) Proliferation assay in S100A4 WT or S100A4 KO Raw264.7 cells using CCK8 assays under indicated cell density in.
Quantification of cell proliferation data collected from three independent experiments. (F) S100A4WT or S100A4KO Raw264.7 cells were activated IFN-γ (20 ng/mL) in combination with LPS (100 ng/mL). Nonactivated macrophages were used as controls. Expression of S100A4 mRNA and protein was analysed by q-PCR (left) and immunoblotting (right), respectively. Band densities (mean ± s.e.m.) for S100A4 were measured in at least three independent immunoblots and normalised to those of β-actin (loading control). Data are presented as mean ± s.e.m. and were analyzed with one-way ANOVA with Tukey's multiple comparisons. The data are from one representative experiment of three independent experiments (B-F). **P value < 0.01, and n.s., not significant.

Supplementary Figure 4
Supplementary Figure 4. Identifying the S100A4 expression in BMDMs. (A) Flow cytometric identification of BMDMs isolated from S100A4 WT or S100A4 KO mice. (B) Flow cytometric analysis the proportion of S100A4 + cells in BMDMs isolated from S100A4 WT-EGFP mice. GMFI, geometric mean fluorescence intensity. (C) BMDMs isolated from S100A4 WT or S100A4 KO mice were activated IFN-γ (20 ng/mL) in combination with LPS (100 ng/mL). Nonactivated macrophages were used as controls. Flow cytometric analysis of the expression of MHCII after stimulation with IFN-γ/LPS for 0 or 12 hours. GMFI, geometric mean fluorescence intensity. Data are presented as mean ± s.e.m. and were analysed by unpaired Student's t-test. The data are from one representative experiment of three independent experiments (A-C). *P value < 0.05, **P value < 0.01, ***P value < 0.001, n.s., not significant. Sections of carcinoma grafts from MCA205 fibrosarcoma cell-bearing S100A4 WT or S100A4 KO male mice were analyzed by immunofluorescence using specific antibody to CD206 (middle).

Supplementary
The immune complexes were detected with a secondary antibody conjugated to Alexa Fluor 555 (red). DNA was stained with DAPI (blue). Scale bars, 200 μm. Immunofluorescent signal corresponding to CD206 was quantitated with Image J. (B) Flow cytometric analysis of the proportion of CD206 + TAMs in the tumour grafts from E0771 breast cancer cell−bearing S100A4 WT or S100A4 KO female mice. Data are presented as mean ± s.e.m and were analyzed with unpaired nonparametric Mann Whitney test. The data are from one significantly changed peptides between S100A4 WT and S100A4 KO BMDMs after 36-hours stimulation with IL4 (20 ng/mL). Gene ontology (GO) term was shown in A, corrected P-value is large than 0.95. KEGG analysis of differential signaling pathways were shown in B. Murine TAMs that isolated from the E0771 breast cancer cell-bearing S100A4 WT-EGFP mice were sorted by flow cytometry into two subset populations: CD45 + F4/80 + CD11b + EGFP + and CD45 + F4/80 + CD11b + EGFP − (left panel). The sorted cells were applied for total RNA extraction and subjected to RNA-sequencing analysis (right panel). (B) q-PCR analysis of gene expression in fatty acid oxidation of S100A4 WT and S100A4 KO BMDMs after 36 hours stimulation with IL4 (20 ng/mL). Supplementary Figure 9. Analysis of STAT6 and PPARs pathways in S100A4 + and S100A4 − macrophages. (A) Immunoblotting analysis of the protein levels of S100A4, STAT6, and phospho-STAT6 in S100A4 WT or S100A4 KO Raw264.7 cells after stimulation with IL4 (20 ng/mL) for indicated times. (B) Immunoblotting analysis of the protein levels of S100A4, STAT6, and phospho-STAT6 in S100A4 WT or S100A4 KO Raw264.7 cells after stimulation with or without IL4 (20 ng/mL) and STAT6 inhibitor (AS1517499, 200 nM) for 36 h. (C) KEGG analysis of PPARs signaling pathways between the S100A4 + and S100A4 -TAMs isolated from E0771 breast cancer cell-bearing mice. The data are from one representative experiment of three independent experiments (A, B).