Unsaturated FAs form lipid accumulation in macrophages

A, Pie chart showing main compositions of free fatty acids in the serum of healthy humans

B, Measurement of oxygen consumption rate (OCR) in macrophages treated with 200μM of PA, SA, OA, LA or control BSA, respectively, under basal conditions or following the addition of oligomycin, FCCP or the electron transport inhibitor Rotenone/antimycin by a seahorse XF-96 analyzer (n=5).

C-E, Macrophages were treated with 200μM of PA, SA, OA, LA or BSA for 4h. Flow cytometric analysis of lysosome, ER and lipid droplet formation by measuring mean fluorescent intensity (MFI) of lysosome tracker (C), ER tracker (D) and BODIPY (E) in macrophages.

F, Multispectral imaging analysis of BODIPY (green), Lysosome (purple), ER (orange), and merged images showing the colocalization status of BODIPY/lyso-tracker and BODIPY/ER-tracker in macrophages treated with PA, SA, OA, LA or BSA at 200μM for 4h.

G, Analysis of lipid droplet formation (red arrow) in macrophages treated with BSA, PA, SA, OA and LA by transmission electron microscope.

Data are shown as mean ± SD in panel B-E (** p ≤ 0.01, *** p ≤ 0.001, **** p≤ 0.0001, as compared to the control BSA group, unpaired Student t test). See also Figure S1.

LA induces the expression of key enzymes of triacylglycerol synthesis in macrophages

A, Key enzymes in the biosynthesis of triacylglycerol.

B-H, Analyzing the expression of genes encoding key enzymes, including Gpam1 (B), Gpam3 (C), Gpam4 (D), Agpat2 (E), Lipin1a (F), Dgat1 (G), Dgat2 (H), in the triacylglycerol biosynthesis pathway in macrophages treated with 400μM of PA, LA or BSA for 4 hours by real-time PCR.

I, Representative confocal images of lipid accumulation by LipidTOX staining (green), expression of GPAT1 (red), DGAT1 () in macrophages treated with BSA, PA and LA (400uM) overnight.

J-M, Quantification of lipid accumulation (J), protein levels of GAPT1 (K) and DGAT1 (M) in macrophages treated with BSA, PA or LA (400μM) overnight.

Data are shown as mean ± SD in panel B-H, J-M (** p ≤ 0.01, *** p ≤ 0.001, **** p≤ 0.0001, ns, non-significant, as compared to the control BSA group, unpaired Student t test). See also Figure S2.

LA induces lipid accumulation through activating the C/EBPα pathway

A, Measurement of C/EBPα gene expression levels in macrophages treated with BSA, PA, or LA (400μM) for 4 hours by real-time PCR.

B, Representative confocal images of C/EBPα protein expression (red) in macrophages treated with BSA, PA, or LA overnight.

C, Quantification of C/EBPα nuclear expression in macrophages treated with BSA, PA, or LA overnight by Image J analysis.

D-J, Real-time PCR analysis of the levels of C/EBPα (D), Gpam1 (E), Dgat1 (F), Dgat2 (G), Cd36 (H), Lipa (I), and Cpt1b (J) in macrophages transfected with 40 nM C/EBPα siRNA or control siNC and then treated with BSA or LA for 4 hours.

K, Measurement of BODIPY fluorescent intensity in C/EBPα-silencing or control macrophages treated with BSA or LA using a fluorescence spectroscopy.

L, Representative confocal images of Oil Red O staining in C/EBPα-silencing or control macrophages treated with BSA or LA (bar, 10μM).

M, Quantification of Oil Red O fluorescence intensity in C/EBPα-silencing or control macrophages treated with BSA or LA.

Data are shown as mean ± SD in panel A, C-K and M (*p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p≤ 0.0001, ns, non-significant, as compared to the control BSA group or control siNC group, unpaired Student t test). See also Figure S3.

FABP4 mediates LA-induced C/EBPα expression in macrophages

A-C, Analysis of the expression of FABP family members, including Fabp3 (A), Fabp4 (B) and Fabp5 (C), in macrophages treated with BSA, PA or LA (400μM) for 4 hours.

D, UMAP of FABP4-positive macrophage subsets using mouse spleen single-cell RNA sequence analysis.

E-J, Violin plots showing relative expression levels of genes, including Fabp4(E), Cd36 (F), PPARγ (G), CEBPA (H), Plin2 (I) and Fabp5 (J) between Fabp4+ vs Fabp4-macrophages indicated in (C).

K, Confocal analysis of FABP4 expression in macrophages treated with BSA, PA or LA (400uM) (bar, 10μM).

L, Comparison of the expression of FABP4 and C/EBPα between FABP4 wildtype (WT) and knockout (KO) macrophages in response to LA treatment (400μM) (bar, 10μM).

M, Realtime PCR analysis of CEBPA expression in WT and FABP4−/− macrophages treated with BSA, PA, and LA (400μM).

Data are shown as mean ± SD in panel A and L (** p ≤ 0.01, *** p ≤ 0.001, as compared to the control BSA group or FABP4−/− group, unpaired Student t test). See also Figure S4.

FABP4 deficiency reduces LA-induced lipid accumulation in macrophages

A-F, real-time PCR analysis of FABP4 (A) and genes encoding key enzymes for triglycerol biosynthesis, including Gpam1 (B), Dgat1 (C), Dgat2 (D), Gpam4 (E), Agpat2 (F) in WT and FABP4 KO macrophages treated with BSA, PA or LA (400μM).

G, Confocal analysis of protein expression of GPAT1 (red), DGAT1 (cyan) and lipid accumulation (LipidTOX staining, green) in LA-treated WT and FABP4 KO macrophages (bar, 10μM).

H-J, Expression levels of GPAT1 (H), DGAT1 (I) and LipidTOX (J) as indicated in panel G were quantified by Image J.

L, Flow cytometric analysis of neutral lipid accumulation as shown by BODIPY staining in WT and FABP4 KO macrophages treated with BSA or LA.

M, Transmission electron microscope showing lipid droplet staining in WT and FABP4 KO macrophages treated with LA.

Data are shown as mean ± SD in panel A-F, H-L (* p≤ 0.01, p** p ≤ 0.01, *** p ≤ 0.001, ns, non-significant as compared to the control BSA group or FABP4−/− group, unpaired Student t test). See also Figure S5.

FAPB4 expression in macrophages promotes lipolysis and breast cancer cell migration

A-D, Realtime PCR analysis of expression of Adrb2 (A), Adcy4 (B), Pnpla2 (C) and Lipe (D) in FABP4 WT and KO macrophages treated with BSA, PA or LA (400μM).

E, Transwell measurement of migration of breast cancer cells cocultured with FA- or BSA-treated FABP4 WT or KO macrophages (Mφ).

F-I, FABP4 WT or KO macrophages were treated with 100μM BSA, PA or LA for 4 hours. Fatty acids in the culture medium were washed away with FBS-free RPMI-1640. Breast cancer cells were added to a transwell and cocultured with these different FA-or BSA-treated FABP4 WT or KO macrophages for 24 hours. The migrated tumor cells were stained and quantified. Migrated MDA-MB-231 cells were shown in panel F and G. Migrated E0771 cells were shown in panel H and I.

J, FABP4 WT and KO macrophages were treated with indicated FAs or BSA for 4 hours. Flow cytometric staining of BODIPY levels in WT and KO macrophages before and after coculture with E0771 tumor cells for 24 hours.

Data are shown as mean ± SD in panel A-D, G, I, J (* p≤ 0.01, p** p ≤ 0.01, *** p ≤ 0.001, ns, non-significant as compared to the control group or FABP4 KO group, unpaired Student t test). See also Figure S6.

High expression of FABP4 in TAMs is associated with more metastasis of breast cancer

A, Comparison of H&E and CD163 staining (brown) between an example of small and large breast cancer tumors in breast cancer patients.

B, Spearman correlation analysis between breast cancer tumor size and CD163+ TAM staining. C, Expression of FABP4 and CD163 was highly correlated as analyzed by the Spearman correlation analysis in breast cancer tissues.

D, Pie chart showing the percentage of breast cancer patients with or without metastasis.

E, Analysis of the staining of H&E, CD163 (brown), FABP4 (red) in primary breast tumors of patients with and without metastasis.

F, Analysis of CD163 expression levels between primary breast tumors of patients with and without metastases.

G, Analysis of FABP4 expression levels between primary breast tumors of patients with and without metastases.

H, Analysis of FABP4 expression levels between alive and deceased breast cancer patients.

I, Scheme of how FABP4 mediates unsaturated FA (yellow)-induced lipid storage and lipolysis in TAMs. When TAMs are exposed to dietary saturated (gray) or unsaturated (yellow) FAs, unsaturated FAs, but not saturated ones, induce FABP4 nuclear translocation and upregulate FABP4 and CEBPA-mediated transactivation of GPAM1 and DGATs, promoting lipid storage as lipid droplets. Once tumor-induced lipolysis occurs, FABP4/unsaturated FAs are secreted from TAMs to induce tumor migration and metastasis.

Data are shown as mean ± SD in panel F-H (p** p ≤ 0.01, *** p ≤ 0.001, *** p ≤ 0.001, unpaired Student t test). See also Figure S7.