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. 2020 Aug 12;217(11):e20191847. doi: 10.1084/jem.20191847

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© 2020 Donadon et al.

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Figure 3. — Morphology as a correlate of lipid metabolism in human CLMs. (A) Representative S-TAMs and L-TAMs sorted from one CLM specimen, cytospun, and stained with Diff-Quick. Scale bar: 20 µm. (B) Volcano plot of the expression profile of L-TAMs and S-TAMs sorted from five CLM specimens. Horizontal dashed line shows FDR (−log10 adjusted P value) 0.05; vertical continuous lines show log2 fold change (logFC) between −1.5 and 1.5. (C) Pathways enriched in L-TAMs versus S-TAMs from five CLM specimens. Z-score is shown on the x axis. For each pathway, the number of genes modulated is reported. (D) GSEA results showing lipoprotein metabolism as a significantly enriched biological process in L-TAMs compared to S-TAMs from five CLM specimens. The green curve represents the enrichment score, showing the measure to which the genes are overrepresented at the top or bottom of a ranked list of genes. Vertical black bars indicate the position in the ranked list of each gene, belonging to the gene set. Genes positioned in the red and blue sides are up-regulated and down-regulated, respectively, in large macrophages compared with small ones. Number of genes in set (genes = 22; FDR = 0.019) and normalized enrichment score (NES = 2.29) are shown. (E) Heatmap representing selected differentially expressed genes (−1.5 < LogFC > 1.5; FDR < 0.05) between L-TAMs and S-TAMs from five CLM specimens. Genes related to cholesterol and lipid metabolism are shown. (F) Representative pictures of immunohistochemical staining of macrophages expressing ApoE (top) and LXR (bottom) in CLM specimens. ApoE is expressed both in hepatocytes (left) and macrophages (right). Scale bars: 50 µm (top panels); 20 µm (bottom panels). (G) Flow cytometry analysis of cholesterol transporters ABCA1, ABCG1, ApoE, and CD36 in L-TAMs and S-TAMs isolated from four CLM specimens. Graphs represent mean ± SEM of four CLM samples analyzed in different experiments. *, P < 0.05 by Mann-Whitney test. (H and I) Lipid content, assessed as MFI of LipidTOX and lipid uptake, obtained as MFI of BODIPY. Bars represent mean + SEM of four different samples. *, P < 0.05 by Mann-Whitney test.