Journal: Cell reports

Article Title: Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1

doi: 10.1016/j.celrep.2024.113837

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Notch4 (A-12) , Santa Cruz , sc-393893;.

Techniques: Virus, Control, Expressing, Plasmid Preparation, Recombinant, Isolation, MTT Assay, Western Blot, Software

Journal: bioRxiv

Article Title: Breast Cancer Macrophage Heterogeneity and Self-renewal are Determined by Spatial Localization

doi: 10.1101/2023.10.24.563749

Figure Lengend Snippet: ( a ) Macrophages plated either directly with oTME cells (cell contact) or separated by transwells for 7 days, followed by 72 hrs of EdU labeling. Macrophages were imaged (mT) at plating and prior to flow cytometry analysis of EdU. Individual dots represent individual fields of view from 3 independent experiments. Data are shown as mean±SD, 2-tailed t-test. Scale bar, 100μm. ( b ) Gene Set Enrichment Analysis of Gene Ontology showing a significant enrichment of Notch signaling pathway in oTME macrophages (GO:0007219; FDR=0.00065; top, enrichment scores; bottom, rank positions of member genes). ( c ) Rosa26 mTmG mice (n=8) with established mammary tumors were treated with Notch4 monoclonal antibodies (15μg/kg body weight, dosed every 3 days, intraperitoneally) or vehicle (PBS) as a control for 20 days. Tumor sizes were recorded by a caliper and shown as mean ±SD, 2-way ANOVA test, Bonferroni-corrected. At the trial endpoint, mammary tumors were imaged (right). ( d ) Macrophage proliferation analysis in mammary tumors from ( c ) following Notch4 treatment (n=6) at study endpoint. Tumors were harvested and scored for Ki67 and CD206 expression in macrophages by flow cytometry. Data are shown as mean±SD, 2-way ANOVA test, Bonferroni-corrected. Macrophage abundance quantified as percent of live CD45+ cells. Data are shown as mean±SD, Mann-Whitney test. ( e ) C57BL/6 female mice were injected with luciferase-expressing HGSOC cells (1 x 10 6 , intraperitoneally) and treated 7 days after tumor BLI signals were established with NOTCH4 or isotype control IgG antibodies (15μg/kg body weight, dosed every 3 days, intraperitoneally). BLI measurements were recorded every week and representative images are presented.

Article Snippet: Then, mice were pooled and randomized into two arms: vehicle-treated (PBS) or Notch4-treated with anti-Notch4 monoclonal antibodies (BioXcell, clone HMN4-14).

Techniques: Labeling, Flow Cytometry, Bioprocessing, Control, Expressing, MANN-WHITNEY, Injection, Luciferase

Journal: bioRxiv

Article Title: Breast Cancer Macrophage Heterogeneity and Self-renewal are Determined by Spatial Localization

doi: 10.1101/2023.10.24.563749

Figure Lengend Snippet: ( a ) Immunoblotting for the cleaved Notch intracellular domain (NICD), ARG1, phospho-PI3K (pAKT), and phospho-MAPK (pERK), confirming active Notch and PI3K pathways in oTME macrophages but not in CSF-1 macrophages. ( b ) Impact of γ-secretase inhibition on proliferation of oTME macrophages. BM monocytes were isolated and plated with oTME cells for 7 days and then were treated with either compound-E (CompE; 10μM) or DMSO as a control for additional 10 days. Macrophages and oTME cells were analyzed by flow cytometry for Ki67 expression. Data (n=3) are shown as mean±SD, Welch’s t- test. ( c ) EdU incorporation in BMDMs following treatment with Adam17 protease inhibitor (A17Pro) or PBS as vehicle control. Cells were plated with oTME cells in the presence of A17Pro or PBS and cultured for seven days. Cell cultures were then labeled with EdU for 48hrs and were analyzed by flow cytometry for EdU incorporation (n=4 replicates). Data are shown as mean±SD, Welch’s t-test. MFI; mean fluorescence intensity. ( d ) Flow cytometry of relative abundances of Ki67+ tumor cells (mT neg CD45 neg ), total immune cells (mT+ CD45 + ), and stromal cells (mT+ CD45 neg ) in control vs. NOTCH4-treated tumors (n=6 replicates). Data are shown as mean ±SD, 2-way ANOVA test, Bonferroni-corrected. ( e ) Immunofluorescence staining of CD31, IBA1, and Ki67 in tumor transplants from (a). Scale bars, 100μm. ( f ) Primary tumor growth (left) and lung metastasis (right) of metastatic TNBC PDX cells (BR18) in NSG mice following NOTCH4 and IgG treatment. Mice were engrafted with 50K cells and treated as in ( c). Growth kinetics and lung dissemination were evaluated by bioluminescence.

Article Snippet: Then, mice were pooled and randomized into two arms: vehicle-treated (PBS) or Notch4-treated with anti-Notch4 monoclonal antibodies (BioXcell, clone HMN4-14).

Techniques: Western Blot, Inhibition, Isolation, Control, Flow Cytometry, Expressing, Protease Inhibitor, Cell Culture, Labeling, Fluorescence, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Breast Cancer Macrophage Heterogeneity and Self-renewal are Determined by Spatial Localization

doi: 10.1101/2023.10.24.563749

Figure Lengend Snippet: ( a ) S patial P r O tein and T ranscriptome S equencing (SPOTS) on tissue from MMTV-PyMT model (n=3 mice). ( b ) Tissue structure. IF staining with EpCAM-PE and CD45-APC to reveal the tissue architecture. Scale bar, 200μm. ( c ) Spatial clustering and ADT signatures of each cluster. Left panel: spatially-informed clusters (1-6) overlaid onto tissue spatial barcodes. Right panel: heatmap of ADT expression for each cluster, where the right colorbar represents the cell-type annotation of each ADT. ( d ) Spatial ADT expression levels of key surface markers for tumor (EpCAM), fibroblasts (PDPN), and macrophages (F4/80, CD86) in fibroblast-enriched region (cluster 2) and adenocarcinoma (cluster 5). Note enrichment of EpCAM high CD86 high expression in adenocarcinoma region (cluster 5; consistent with TNMs) vs. enrichment of PDPN high F4/80 high expression in fibroblasts-enriched regions (cluster 2; consistent with SAMs). ( e ) Spatial correlation of lineage (NK1.1, CD4, CD8, CD11b, F4/80, MHC-II, EpCAM) immunostimulatory (CD27, CD86, CCR2, CD11c), and immunosuppressive (PD-L1, Sca-1) ADTs in fibroblast-enriched region (cluster 2) and adenocarcinoma region (cluster 5). ADTs are colored by bivariate Moran’s I (color scale). The size of each dot represents the inverse of standard error of the mean (S.E.M; n=3 mice). Dendrograms indicate the hierarchical clustering of the ADTs. ( f ) EpCAM and CCR2 vs. PDPN and F4/80 ADTs co-expression levels overlaid onto tumor sample A tissue (Methods) and immunophenotyping of SAMs and TNMs. Middle panel: boxplots of EpCAM and CCR2 vs. PDPN and F4/80 co-expression levels (addition of EpCAM and CCR2, or PDPN and F4/80 expression values). Right panel: violin plots of immune-stimulatory and suppressive ADT expression levels. Kolmogorov–Smirnov test, * P < 0.05, ** P < 0.01, *** P < 0.001, otherwise not significant (n.s). Each boxplot ranges from the first and third quartiles with median values shown as middle lines, and the whiskers represent 1.5 times the interquartile range. ( g ) Volcano plot showing log fold changes (logFC) of top 5,000 most variable genes between fibroblast-enriched region (cluster 2) vs. adenocarcinoma region (cluster 5) and their significance (y axis; -log10 scale). Genes are dotted and colored by logFC levels (color scale). The size of each dot represents the difference in the fraction of detection between the two groups. Macrophage-related genes are annotated. P -values were determined by Wilcoxon Rank Sum test. Vertical dotted lines represent ±0.2 logFC. Horizontal dotted lines represent FDR of 0.05 (-log10 scale). Raw, FDR corrected P -values, and logFC values are listed in Supplementary Table 10. ( h ) Gene Set Enrichment Analysis of scRNA-seq Ly6a +SAM gene signature (Supplementary Table 7; ) in the fibroblast-enriched region (cluster 2) ( P = 0.002). ( i ) Gene expression of IFNα response genes ( Ly6a, Ly6c1, Ifit1, Ifit3, Ifitm3, Irf7, Isg15, Stat1, Stat2 ) in SAMs (cluster 2) and TNMs (cluster 5). Genes are dotted and colored by expression levels. The size of each dot represents the percentage of expression in the tissue area. ( j ) Notch signaling pathway activity in fibroblast-enriched regions (SAMs) and adenocarcinoma regions (TNMs) across all three biological replicates. Left panel: violin plots of NOTCH4 ADT expression levels. Right panel: violin plots of Notch signaling pathway (GO:0007219; n=179 genes) transcriptional expression levels. Kolmogorov–Smirnov test, * P < 0.05, ** P < 0.01, *** P < 0.001, otherwise not significant (n.s).

Article Snippet: Then, mice were pooled and randomized into two arms: vehicle-treated (PBS) or Notch4-treated with anti-Notch4 monoclonal antibodies (BioXcell, clone HMN4-14).

Techniques: Staining, Expressing, Gene Expression, Activity Assay

Journal: bioRxiv

Article Title: Intestinal microbiota programming of alveolar macrophages influences severity of respiratory viral infection

doi: 10.1101/2023.09.21.558814

Figure Lengend Snippet: (A) Schematic design for ex vivo studies of AM. (B) Comparison of basal SFB + and SFB - AM gene expression by RNA-seq, shown as a volcano plot. (C) SFB + and SFB - AM were exposed to UV-CA09 with or without Notch4 neutralizing, or isotype control antibodies. RNA was harvested 24 hours later, and gene expression assayed by RT-qPCR. Results are a heat map summary with full results shown in FS7. (D) AM and CA09 stocks were incubated at 37C, or indicated temperature, for 45 minutes and supernatant were collected. In addition, some cells had new media added and were incubated for another 24 hours. (i) virus infection titers in supernatant at 45 minutes and 24 hours were assayed. (ii) Supernatant were collected after 45 minutes incubated with CA09 and viral genomes quantitated by RT-qPCR. (iii) Cell-free supernatant of 15 hours AM cultures were incubated with live CA09 stocks, with or without C1qa-neutralization or isotype antibody for 45 minutes, at which point virus infection titers were assayed. (iv) Cell lysates were generated upon CA09 removal or 24 hours later and viral genomes quantitated by RT-qPCR. All experiment n = 4-5 mice per group. Data is representative of two independent experiments, yielding an identical pattern of results. Statistical analysis: One-way ANOVA. *p<0.05, **p<0.01,, ****p<0.0001, ns not significant.

Article Snippet: After sorting, cells were treated with either isotype control antibody (Cat# BE0091, BioXCell) or an anti-Notch4 monoclonal antibody (20μg/mL) (Cat# BE0129, BioXCell), known to neutralize Notch 4 [ ] for 30 minutes.

Techniques: Ex Vivo, Comparison, Gene Expression, RNA Sequencing, Control, Quantitative RT-PCR, Incubation, Virus, Infection, Neutralization, Generated

Journal: bioRxiv

Article Title: Intestinal microbiota programming of alveolar macrophages influences severity of respiratory viral infection

doi: 10.1101/2023.09.21.558814

Figure Lengend Snippet: (A) As schematized, AM was isolated from SFB - and SFB + mice and treated with UV-inactivated CA09 in the presence/absence of anti-Notch4 monoclonal antibodies. qRT-PCR results of Il6, and type I interferon-related genes from AM. (B) (i) As schematized, CD45.1 SFB - mice were administered SFB or PBS. Seven days later, AMs were isolated by FACs. Some SFB - AM were then treated with an anti-Notch4 monoclonal antibody or isotype. AM were then transferred to CD45.2 SFB - mice, 5×10 5 per mouse. One day post transfer, mice were inoculated with CA09. Mice were euthanized on day 4.5 post virus inoculation. FACs graphical representation, frequencies, and cell numbers of endogenous and exogenous AM. (ii) lung CA09 viral genomes copies, survival rate and body weight. (iii) FACs graphical representation and frequencies of AECs-containing CA09. All experiments n =5 mice per group. Data is representative of two independent experiments, yielding an identical pattern of results. Statistical test: Relative expression of pro-inflammatory genes, CA09 RNA copies, frequencies of GFP+ cells data: one-way ANOVA. Survival data; one-way ANOVA. Body weight: Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns not significant.

Article Snippet: After sorting, cells were treated with either isotype control antibody (Cat# BE0091, BioXCell) or an anti-Notch4 monoclonal antibody (20μg/mL) (Cat# BE0129, BioXCell), known to neutralize Notch 4 [ ] for 30 minutes.

Techniques: Isolation, Bioprocessing, Quantitative RT-PCR, Virus, Expressing