Journal: Science Advances

Article Title: Macrophages orchestrate the expansion of a proangiogenic perivascular niche during cancer progression

doi: 10.1126/sciadv.abg9518

Figure Lengend Snippet: ( A and B ) Box and whisker plots (A) and scatter plot (B) showing normalized mean M1- and M2-associated gene scores across the indicated TAM clusters identified using scRNA-seq. ( C and D ) Subset unique, significantly up-regulated GO terms (C) and individual genes (D) between the two subsets of protumoral TAM. ( E ) FACS-gated live [7-aminoactinomycin D–negative (7AAD − )] F4/80 hi TAMs from enzyme-dispersed MMTV-PyMT tumors separated on the basis of CD206 and MHCII expression (left) and assessed for Lyve-1 expression (right; color-shaded histograms) against that of the fluorescence minus one staining (FMO) control (open black line). ( F ) Quantification of the gated populations in (E) ( n = 4 tumors). ( G ) PCA plot of the 2000 most variable genes from the bulk-sequenced TAM populations ( n = 5 tumors), using CD206 − and MHCII − TAMs as a comparator. ( H ) Heatmaps comparing the relative expression of selected differentially expressed genes identified in the scRNA-seq (left) and bulk RNA-seq (right); population color is indicative of the populations identified in (G). ( I ) Representative image of a frozen section of MMTV-PyMT tumor showing DAPI (nuclei; blue); intravenous dextran marking vasculature (green), F4/80 (magenta), and Lyve-1 (red); and colocalizing pixels for Lyve-1 and F4/80 (white); scale bars, 25 μm. ( J to M ) Schematic for experimental approach to label pvTAMs using Dil-labeled liposomes (J). (K) Representative images of frozen sections of MMTV-PyMT tumors showing DAPI (nuclei; blue); intravenous dextran marking vasculature (green), Dil (red), and F4/80 (magenta); and Dil/F4/80 colocalizing pixels (white) (right panel alone); scale bars, 25 μm (left) and 50 μm (right). (L) Quantification of the spatial location of Dil + F4/80 + TAMs ( n = 5 mice). (M) Analysis of the surface phenotype of Dil +/− TAM from enzyme-dispersed tumors within the F4/80 + gate. Box and whisker plots; boxes show median and quartiles. Bar charts represent mean, and the dots show individual tumors and mice. **** P < 0.0001.

Article Snippet: The following antibodies against the indicated antigen were purchased from Thermo Fisher Scientific and were used at 1 μg/ml unless stated otherwise: CD3ε allophycocyanin (APC) and phycoerythrin (PE) (145-2C11), CD4 FITC (RM4-5), CD8β eFluor 450 (H35-17.2), CD11b APC–eFluor 780 (M1/70), CD11b BV510 (M1/70), CD11c APC (N418), CD16/32 (2.4G2; Tonbo Biosciences), CD19 APC (6D5; BioLegend), CD29 APC (eBioHMb1-1), CD31 eFluor 450 and PE (390), CD34 FITC and APC (RAM34), CD45 APC–eFluor 780, FITC, and peridinin chlorophyll protein (PerCP)–Cy5.5 (30-F11), CD90.2 eFluor 450 (53-2.1), CD90.1 eFluor 450 (HIS51), CD90.1 BV510 (OX-7), CD103 PE (2E7), CD206 APC (FAB2535A; Bio-Techne), F4/80 PE (BM8; BioLegend), F4/80 BV421 (BM8; BioLegend), FAP (10 μg/ml; AF3715, Bio-Techne), Ly6C PE and eFluor 450 (HK1.4), Ly6G FITC (1A8; BioLegend), Lyve-1 Alexa Fluor 488 (ALY7), MHCII PE, FITC, and eFluor 450 (M5/114.15.2), NG2 Alexa Fluor 488 (AB5320A4; Millipore), NK1.1 APC (PK136), PDGFRα APC and PerCP-Cy5.5 (APA5), PDGFRβ PE (APB5), and Ly6A/E Alexa Fluor 700 (D7).

Techniques: Whisker Assay, Expressing, Fluorescence, Staining, RNA Sequencing Assay, Labeling, Liposomes

Journal:

Article Title: IL-4/IL-13-Dependent Alternative Activation of Macrophages but Not Microglial Cells Is Associated with Uncontrolled Cerebral Cryptococcosis

doi: 10.2353/ajpath.2009.080598

Figure Lengend Snippet: In the presence of IL-4 and IL-13, macrophages but not microglial cells expressing markers of alternative activation are detected in the brains of susceptible BALB/c wild-type mice at 60 d.p.i. A: Wild-type (WT) mice show that large pseudocystic lesion, harboring many small yeasts and yeast fragments, is bordered by inflammatory leukocytes, consisting mainly of large rounded foamy macrophages (black arrowheads). H&E staining, magnification = original ×600. The large foamy rounded cells are identified as MHC class II+ macrophages tightly bordering the lesion. Activated MHC class II+ ramified microglial cells (white arrowheads) are demonstrated in the outer vicinity of the lesion. Anti-MHC class II immunostaining, magnification = original ×600. Large round cells strongly express the CD206 antibody, whereas the antibody against the mannose receptor did not detect ramified microglial cells. Anti-CD206 immunostaining; magnification = original ×600. Many round cells, but not microglial cells, bordering the lesion strongly express the YM1 antibody. Anti-YM1 immunostaining; magnification = original ×600. B: Upper panel: Ultrastructural analysis of the identified type of macrophages reveals that they harbor yeasts and yeast fragments within their cytoplasm, and that they also contain numerous small vacuoles corresponding to their ‘foamy’ appearance, which are phagolysosomes. Lower panel: The ultrastructural analysis of microglial cells identifies a cell with dense-staining nucleus and marginated chromatin free of cryptococci or vacuolated lysosomes. Electron microscopy; magnification = original ×3000.

Article Snippet: Additionally, CD206 (mannose receptor) rat anti-mouse antibody (Serono, Unterschleißheim, Germany) and YM1 (ECF-L) goat anti-mouse antibody (R&D Systems, Minneapolis, MN) were used for staining of alternatively activated macrophages.

Techniques: Expressing, Activation Assay, Staining, Immunostaining, Electron Microscopy

Journal:

Article Title: IL-4/IL-13-Dependent Alternative Activation of Macrophages but Not Microglial Cells Is Associated with Uncontrolled Cerebral Cryptococcosis

doi: 10.2353/ajpath.2009.080598

Figure Lengend Snippet: Alternatively activated macrophages develop in the CNS of susceptible wild-type, and IL-13T/+ mice after i.n. infection with C. neoformans. A–E: Mannose receptor (CD206) expression as a marker for alternative activation of macrophages in the brain (60 dpi). In wild-type (WT) and IL-13T/+ mice, macrophages show a strong expression of CD206, whereas the receptor is absent on cells of IL-4Rα−/− (B), IL-4−/− (C), and IL-13−/− (D) mice. In no case were microglial cells CD206 positive. Anti-CD206 immunostaining, magnification = original ×400. F–J: Chitinase YM1 expression as a marker for alternative activation of myeloid leukocytes in the brain (60 dpi). In wild-type and IL-13T/+ mice, macrophages show a strong expression of YM1, whereas the molecule is absent in cells of IL-4Rα−/− (G), IL-4−/− (H), and IL-13−/− (I) mice. In no case microglial cells were YM1 positive. Anti-YM1 immunostaining; magnification = original ×400.

Article Snippet: Additionally, CD206 (mannose receptor) rat anti-mouse antibody (Serono, Unterschleißheim, Germany) and YM1 (ECF-L) goat anti-mouse antibody (R&D Systems, Minneapolis, MN) were used for staining of alternatively activated macrophages.

Techniques: Infection, Expressing, Marker, Activation Assay, Immunostaining