Fig 2: Validation of prognostic macrophage markers. A-C, Representative immunofluorescence staining of breast cancer TMAs with MF markers CD163/CD68 (green) and CD206 (yellow), together with PLAC8 (magenta), SERPINH1 (blue), MORC4 (red) (A, B), or MHCII (red) (C). Nuclei were counterstained with DAPI (white). Magnifications serve to illustrate the presence of CD206+ MORC4+ (A, upper panels), CD206+ SERPINH1+ (B), and CD206- MHCII+ macrophages (C). Furthermore, SERPINH1+ PLAC8+ fibroblasts are displayed (A, lower panels)

Fig 3: Quantitative analysis of immunofluorescence staining. A-C, I, J and M, Analysis of TMA staining for the abundance of all macrophages (A) and the CD206+ (B) and CD206- (C) subsets in normal tissue (n = 5), DCIS (n = 16) and invasive breast cancer (n = 154). Abundance of CD206+ MORC4+ (I), CD206+ SERPINH1+ (J), and CD206- MHCII+ (M) macrophages was also analyzed in the same tissues. P-values were calculated using one-way ANOVA with Dunn's multiple comparison test. *P < .05. D-H, K, and L, Kaplan-Meier estimates analyze the association of indicated cell populations with patient survival. Q1 marks the lowest quartile of indicated cell subtype abundance in tissues and Q4 the highest quartile (n = 38 in each quartile for D-G, H, and K; n = 24 for L). Hazard ratios were calculated between Q1 and Q4

Fig 4: iCSFCs have fibroblast properties. (A) Gene expression of fibroblast cell markers was examined by RT-PCR. Most fibroblast genes were expressed in iCSFCs, with the exception of Acan, MMP13 and Sox6. GAPDH was used as an internal control. (B,C) Immunocytochemistry analysis of iCSFCs for fibroblast marker proteins. Immunostaining was used to indicate fibroblast markers (red) in CFF-3 cells (B) and iCSFCs (C), including ALCAM, Col1A1, HSP47 and S100A4. Cell nuclei were labeled with DAPI (Scale bars, 50 µm).