Fig 2: The effects of MTL-CEBPA treatment on TAMs in patients with HCC. A, Representative images of a CR patient's biopsies with a complete loss of protumoral M2 macrophages (blue squares: CD68+ CD163+ CD64− cells) are shown in bottom (CR posttreatment) panel when compared with top (CR pretreatment) panel. White squares represent the pan-macrophage population expressing CD68+. Pseudo-color image: created by virtual slides alignment and imported in Halo software for biomarker analysis. B, A heat map of macrophage subsets was set up based on log2 fold change between pre- and posttreatment (cell densities) samples of 3 HCC patients including the CR as shown in A, stable disease (SD), and progressive disease (PD). The macrophage populations analyzed were: pan-macrophage (CD68+ cells), antitumoral M1 macrophages (CD68+CD64+CD163−CD206− cells), activated M1 macrophages (CD68+CD64+CD163−CD206− cells), protumoral M2 macrophages subset 1 (CD68+CD163+CD64− cells), protumoral M2 macrophages subset 2 (CD68+CD206+CD64− cells), protumoral M2 macrophages subset 3 (CD68+CD163+CD206+CD64− cells), and activated M2 subsets characterized by IL10 production.

Fig 3: Docosahexaenoic acid (DHA) treatment modifies macrophage subsets in pediatric NAFLD.A) Immunohistochemistry for CD68 in pediatric NAFLD biopsies. The number of portal (red arrowheads) CD68+ macrophages (MΦs) is reduced after DHA treatment (T1) in comparison with baseline (T0) biopsies. No modifications in lobular CD68+ MΦ number (yellow arrows) are observed at T1. Original Magnification (OM) = 10x. B) Immunohistochemistry for S100A9 in pediatric NAFLD biopsies. The number of S100A9+ macrophages is reduced after DHA treatment (T1) in comparison with baseline (T0) biopsies. C) Double immunofluorescence for CD206 and CD68 in pediatric NAFLD biopsies. CD206+ macrophages are increased (yellow arrows) after DHA treatment (T1) in comparison with baseline (T0) biopsies. Original Magnification (OM) = 10x.

Fig 4: Senescent tumor cells inhibit CD8+ T cell activation via monocytes to M2 type macrophage differentiation. A) CD8+ T cells activation in p16INK4A positive and negative CRC. p16INK4A negative and positive CRC were dissected serially and immunostained with p16INK4A/CD8, Ki67/CD8 and Tim3, and the percentage of Ki67+/CD8+ cells was analyzed. Three randomly selected areas of the tumor tissue were photographed (200×) and then analyzed for Ki67+/CD8+ cell infiltration and Tim3+ cell number; the results were averaged and then presented as a dot graph. The p value was calculated by the Mann–Whitney U test. B) CD68 and SA‐β‐Gal staining analysis in CRC. C) Macrophage distribution analysis with CD206 in p16INK4A positive or negative CRC. The number of CD206 positive macrophages in CRC according to the grades of p16INK4A immunostaining was presented as a dot graph. D) T cell proliferation assay. Isolated primary monocytes were incubated with CM from SW480 or SW480/ROS for 7 days and then monocyte differentiation was analyzed (left lower panel). CFSE labeled CD8+ T activation by anti‐CD3/CD28 beads was performed under coculture with differentiated macrophages for 96 h, and suppression of T cell proliferation was measured by flow cytometry. The p value was calculated by one‐way ANOVA and post hoc analysis C,D). E) Microdissection analysis. The expression in RNA sequencing indicates the relative values of the p16INK4A positive region compared with the p16INK4A negative region. F) CRC tissues were serially sectioned for p16INK4A and CSF1 immunostaining. The p value was calculated using the χ 2 test. G) CSF1 polarized monocytes to M2 macrophages. Isolated primary monocytes were cocultured with SW480 (control, H2O2 treated, CSF1 overexpressing, and CXCL12 overexpressing) for 6 days and then analyzed for cell morphology (upper panel), and CD206 expression by FACS (lower panel), respectively. N and n indicated the number of cases and independent experiments, respectively.

Fig 5: The process of macrophage–myofibroblast transition in proliferative vitreoretinal disorders. Based on our results, we propose the depicted sequence of changes in monocytes while they transition to myofibroblasts. First, macrophages acquire an M2 phenotype, and, at this stage, the expression of corresponding markers, such as CD206, is enhanced. Subsequently, myofibroblast-specific genes are turned on, and apparently, α-SMA and FSP-1 become detectable, whereas the expression of FAP-α remains precluded, as the latter marker was not costained with CD68. The figure is generated using BioRender.com.