human breast cancer tissue microarray sections  (Biomax Inc)

Journal: Nature Communications

Article Title: Single cell atlas identifies lipid-processing and immunomodulatory endothelial cells in healthy and malignant breast

doi: 10.1038/s41467-022-33052-y

Figure Lengend Snippet: a Sankey diagram (left panel), showing the scmap cluster projection of annotated (peri-) tumoral breast-derived ECs (left part; n = 8433 endothelial cells) to subclusters of the EC taxonomy in non-small cell lung carcinoma (right part) and box plots (right panel) depicting the scmap similarity index. PCV post-capillary venules, EC endothelial cell, LS lower sequencing depth. Boxes extend from the 25 th to 75 th percentiles, line in the middle of the box is plotted at the median. Whiskers = min and max. b Three-dimensional principal component (PC) analysis on the pairwise Jaccard similarity coefficients of marker genes between EC subtypes in lung and breast. Color coding according to EC subtypes (lung – squares; breast – circles). c Venn-diagrams of the top-50 marker genes in the indicated EC subtypes in lung and breast ECs. Numbers in the middle reflect genes congruently ranking in the top-50 in both tissues. d Heatmap of the expression levels of the indicated immunoregulatory genes in the different breast EC subtypes. Venous EC subtypes are indicated by dashed lines. Color scale: red – high expression, blue – low expression. e Quantification of HLA-DR signal in ACKR1 + CD105 + pNEC and TEC represented as a percentage of the CD105 + vessel area. Data are mean ± SEM, n = 7, * p < 0.05 (exact p -value = 0.0045), paired t-test (two-tailed). f Representative micrographs of human breast peri-tumoral (left) and tumoral (right) tissue sections, immunostained for CD105 and HLA-DR, stained for ACKR1 by RNAscope and counterstained with Hoechst ( n = 7). Brightness was decreased linearly (gamma = 1) to improve visibility for ACKR1 and CD105. Scale bar: 10 µm.

Article Snippet: Formalin-fixed paraffin-embedded human breast tissue sections were subjected to RNAscope in situ hybridization using the RNAscope Multiplex Fluorescent v2 assay (ACDBio) combined with immunofluorescence—Integrated Co-Detection Workflow according to the manufacturer’s instructions (Pretreatment and RNAscope Multiplex Fluorescent v2 Assay according to protocol 323100-USM and MK-5150).

Techniques: Derivative Assay, Sequencing, Marker, Expressing, Two Tailed Test, Staining, RNAscope

Journal: Nature Communications

Article Title: Single cell atlas identifies lipid-processing and immunomodulatory endothelial cells in healthy and malignant breast

doi: 10.1038/s41467-022-33052-y

Figure Lengend Snippet: a UMAP-plot of T-/NK cells color coded by subcluster. NK natural killer. b Heatmap of the expression levels of canonical marker genes of T-/NK cell (sub-)types. Color scale: red – high expression, blue – low expression. c UMAP-plot of myeloid cells color coded by subcluster. TR tissue resident, LS lower sequencing depth. d Heatmap of the expression levels of canonical genes in myeloid cells. Color scale: red – high expression, blue – low expression. TAM tumor associated macrophages. e Schematic overview of the receptor ligand interaction analysis. Clusters containing <100 cells: mast cells, plasma cells and plasmacytoid dendritic cells. EC endothelial cell, LFC log fold change, RLI receptor ligand interaction. f Circos plots representing RLI analysis between angiogenic/venous ECs and immune cells. Receptor is expressed on immune cell subclusters, ligand is expressed on angiogenic ECs (left panel) or venous ECs (right panel). Plots are color coded for the receptor–ligand pairs (arrows, gene names) and immune cell subclusters expressing the receptor (bars perpendicular to inner circle). Previously unknown RLI pairs between ECs and specific immune cell subtypes are indicated in bold (genes) and with asterisks (subclusters). g Representative micrographs of human breast tumoral tissue sections, immunostained for CD105 and CLEC2B (left panels) or CD16 and KLRF1 (right panels) and counterstained with Hoechst ( n = 8). Middle panels: magnifications of the white boxed areas in the upper panels. Bottom panels: magnifications of the orange boxed areas in the upper panels. Dotted white line indicates a CLEC2B + blood vessel, dotted orange lines indicate KLRF1 + NK cells in the vicinity of the blood vessel. Scale bar: 50 µm.

Article Snippet: Formalin-fixed paraffin-embedded human breast tissue sections were subjected to RNAscope in situ hybridization using the RNAscope Multiplex Fluorescent v2 assay (ACDBio) combined with immunofluorescence—Integrated Co-Detection Workflow according to the manufacturer’s instructions (Pretreatment and RNAscope Multiplex Fluorescent v2 Assay according to protocol 323100-USM and MK-5150).

Techniques: Expressing, Marker, Sequencing, Clinical Proteomics

Journal: Nature Communications

Article Title: Single cell atlas identifies lipid-processing and immunomodulatory endothelial cells in healthy and malignant breast

doi: 10.1038/s41467-022-33052-y

Figure Lengend Snippet: a Waterfall plot of top-15 up- and downregulated metabolic pathways in metabolic gene set enrichment analysis in TECs compared to pECs (gray – up in pEC, red – up in TEC). Asterisks mark gene sets involved in lipid metabolism. b Volcano plot showing differential metabolic gene expression analysis of pECs versus TECs. Key pEC-enriched marker genes involved in lipid metabolism are indicated. Gray, significant (adjusted p-value (Benjamini–Hochberg) < 0.05); dark blue, not significant. Differential expression analysis was performed using limma , the magnitude of differential expression (log2 fold change) and false discovery rate adjusted p -values (Benjamini–Hochberg) are provided on the x - and y -axis, respectively. c Dot plot heatmap of the gene expression levels within the LIPEC signature in breast EC subclusters. The color intensity of each dot represents the average level of marker gene expression, while the dot size reflects the percentage of cells expressing the marker within the subcluster. Color scale: red – high expression, blue – low expression. LIPEC lipid processing EC, TF transcription factor, LS lower sequencing depth. d Quantification of the FABP4 + CD105 + vessel area in peri-tumoral and tumoral breast tissue. Data are mean ± SEM, n = 7, ** p < 0.01 (exact p -value = 0.0014), two-tailed paired t-test. For a representative image of the stained peri-tumoral - tumor border, see Supplementary Fig. . e Dot plot heatmap of the expression of PPARG and LXRA (left panel) and their respective regulons from SCENIC analysis (right panel). The color intensity of each dot represents the average level of gene (left) or regulon (right) expression, while the dot size reflects the percentage of cells expressing the gene/regulon within the cell subcluster. Color scale: red – high expression, blue – low expression. f Quantification of the % of EC nuclei with positive PPARG staining by RNAscope in CD105 + FABP4 + vessels vs . CD105 + FABP4 − vessels in human breast tissue (tumor and peri-tumoral tissue pooled per patient). Data are mean ± SEM, n = 8, **** p < 0.0001 (exact p -value < 0.0001), two-tailed paired t-test. g Representative micrographs of human breast tumor tissue sections, immunostained for CD105 and stained for FABP4 and PPARG by RNAscope and counterstained with Hoechst ( n = 8). Right panels: magnifications of the boxed areas in the middle panels. Red arrows point to PPARG transcripts stained by RNAscope. Scale bar: 10 µm.

Article Snippet: Formalin-fixed paraffin-embedded human breast tissue sections were subjected to RNAscope in situ hybridization using the RNAscope Multiplex Fluorescent v2 assay (ACDBio) combined with immunofluorescence—Integrated Co-Detection Workflow according to the manufacturer’s instructions (Pretreatment and RNAscope Multiplex Fluorescent v2 Assay according to protocol 323100-USM and MK-5150).

Techniques: Gene Expression, Marker, Quantitative Proteomics, Expressing, Sequencing, Two Tailed Test, Staining, RNAscope

Journal: Nature Communications

Article Title: Single cell atlas identifies lipid-processing and immunomodulatory endothelial cells in healthy and malignant breast

doi: 10.1038/s41467-022-33052-y

Figure Lengend Snippet: a Schematic overview of the survival analysis in the retrospective clinical cohort and immunostaining validation. UH university hospital, BC breast cancer, HER2 human epidermal growth factor receptor 2, BMI body mass index, HR hormone receptor status. Color coding in the clinical characteristics panel reflects differences in age, BMI, tumor stage & grade (Supplementary Data ). Color coding underneath the treatment stratification panel indicates patients that did (green) or did not (blue) receive metformin treatment during follow up. b Cumulative incidence function estimate of BC-specific survival (left panel) and the distant relapse free interval (right panel) in BC patients stratified by intake of a metformin. Color coded by group: blue – control, green – patients treated with metformin, purple – control matched for age, BMI, tumor stage & grade and hormone receptor status. P -values were calculated by the Kaplan–Meier (log rank) test between metformin therapy and without metformin therapy groups (blue for unmatched control patients; purple for matched control patients). Numbers in the boxes underneath the curves depict the number of patients per group that are at risk for the event (mortality in left panel, mortality/development of metastasis in right panel) at the indicated time points. c Quantification of FABP4 + blood vessels (% area of total CD105 + blood vessels) in non-diabetic BC patients ( n = 8) and in diabetic patients without ( n = 8) or with ( n = 9) metformin treatment. Data are mean ± SEM, ** p < 0.01 (exact p -values = 0.0030 and 0.0023, respectively), one-way ANOVA followed by Dunnett’s multiple comparisons test. d Representative micrographs of human breast tumor tissue sections in control non-diabetic (left; n = 8) or diabetic (middle; n = 8) control BC patients and in (diabetic) BC patients treated with metformin (right; n = 9), immunostained for CD105, FABP4 and counterstained with Hoechst. Arrowheads denote CD105 + FABP4 + vessels, asterisks denote (putative) adipocytes, which (besides LIPECs) are also positive for FABP4. Brightness was increased linearly (gamma = 1) to improve visibility for CD105 and FABP4. Scale bar: 75 µm.

Article Snippet: Formalin-fixed paraffin-embedded human breast tissue sections were subjected to RNAscope in situ hybridization using the RNAscope Multiplex Fluorescent v2 assay (ACDBio) combined with immunofluorescence—Integrated Co-Detection Workflow according to the manufacturer’s instructions (Pretreatment and RNAscope Multiplex Fluorescent v2 Assay according to protocol 323100-USM and MK-5150).

Techniques: Immunostaining, Biomarker Discovery, Control