Journal: Advanced Science

Article Title: Melanoma Derived Exosomes Amplify Radiotherapy Induced Abscopal Effect via IRF7/I‐IFN Axis in Macrophages

doi: 10.1002/advs.202304991

Figure Lengend Snippet: CircPIK3R3/IRF7/I‐IFN axis participates in the combination of radiotherapy and anti‐PD1 mediated abscopal effect. A) The treatment model involving RT, anti‐PD1, and RO8191: C57/BL6 mice were subcutaneously inoculated with 1 × 106 sh‐NC B16F10 cells or sh‐circ‐0011074 B16F10 cells. On day 5, C57/BL6 mice were intravenously injected with 1 × 106 B16F10‐luc cells. Starting from day 6, mice were administered the IFN receptor agonist RO8191 via daily intraperitoneal injections at a dose of 1 mg kg −1 . On day 7, radiotherapy was initiated, with a daily dose of 8 Gy administered for 3 consecutive days. On day 7, mice were also administered anti‐PD1 via intraperitoneal injection every 2 days at a dose of 100 µg/mouse until the endpoint of observation. B,C) Measurement of subcutaneous tumor weight in each group to assess treatment efficacy ( n = 3). D,E) Evaluation of fluorescent intensity in lung metastatic foci using bioluminescence imaging to assess treatment efficacy ( n = 6). F,G) Immunohistochemical examination of CD8 + T cell infiltration in subcutaneous tumors and lung metastatic foci ( n = 3). H,I) Immunofluorescence detection of IRF7 + macrophage infiltration in subcutaneous tumors and lung metastatic foci (n = 3). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by two‐tailed unpaired Student t‐test.

Article Snippet: Anti‐mouse PD‐1 mAb (Bio X Cell, USA) was administered intraperitoneally (200 mg per mouse) every 2 days during treatment.

Techniques: Injection, Imaging, Immunohistochemical staining, Immunofluorescence, Two Tailed Test

Journal: Biomolecules

Article Title: Hypoxia-Induced Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Regulate Macrophage Polarization and Enhance Angiogenesis to Promote Diabetic Wound Healing

doi: 10.3390/biom15111504

Figure Lengend Snippet: Anti-inflammatory and pro-angiogenic effects of hy-EVs in vitro. ( a ) Immunofluorescent staining of CD86 (red) and CD206 (green) and ( b ) quantitative analysis of their fluorescence intensities ( n = 3 per group). ( c ) ROS fluorescent staining of HSFs and HUVECs in different treatment groups and ( d ) quantitative analysis of their fluorescence intensities ( n = 3 per group). ( e ) Immunofluorescence staining of HIF-1α (green), VEGFA (green) and CD31 (red) expression in different treatment groups, and the nuclei were stained with DAPI (blue); ( f ) quantitative analysis of their fluorescence intensities ( n = 3 per group). ( g ) Western blot analysis of the expression levels of HIF-1α, VEGFA and CD31 in different treatment groups (original Western blot images can be found in ), and ( h ) their quantitative analysis ( n = 3 per group). Data are mean ± SD, *** p < 0.001. All p values were obtained by one-way ANOVA.

Article Snippet: N-EVs and hy-EVs were labeled with Cell Plasma Membrane Red Fluorescence Staining Kit (PKH26, Beyotime, Shanghai, China).

Techniques: In Vitro, Staining, Fluorescence, Immunofluorescence, Expressing, Western Blot

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Representation and validation of the AF4‐MALS‐FLD method . (A) Overview of the workflow used for identification of EV surface proteins. PE‐conjugated antibodies were incubated with the sample (e.g. pre‐purified EVs, cell culture supernatant, urine, or plasma) and loaded into the AF4 channel. (B) The light scatter elution profile (in relative scale) (black, full line), UV elution profile (black, dotted line) and the size determination ( R rms in nm) (red) obtained by the multi‐angle light scattering (MALS) detector is plotted against time for labelling of SK‐BR‐3‐derived EVs with PE‐conjugated anti‐CD81 antibody. (C) The fluorescent light detector (FLD) signal (in relative scale) for SK‐BR‐3‐derived EVs labelled with PE‐conjugated anti‐CD9, anti‐CD63 and anti‐CD81 is plotted against time. (D) Transmission electron microscopy (TEM) images of different fractions of the AF4‐MALS‐FLD elution profile are shown (scale bar = 200 nm).

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Incubation, Purification, Cell Culture, Clinical Proteomics, Multi-Angle Light Scattering, Derivative Assay, Transmission Assay, Electron Microscopy

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: AF4‐MALS‐FLD analysis of EV surface proteins with biomarker potential in prostate and breast cancer . MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. (A) The elution profile (in relative scale) of the multi‐angle light scatter (MALS) detector and the size ( R rms in nm) were plotted against time. The fluorescent light detector (FLD) signal for MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs labelled with (B) PE‐conjugated anti‐EpCAM and (C) PE‐conjugated anti‐HER2 antibodies were plotted. (D) From FLD elution profiles, the area under the curve for the EV peak (24–80 min) was determined. Unstained EV samples were used as a negative control. (E) Different concentrations (6 × 10 9 , 8 × 10 9 , 1 × 10 10 and 2 × 10 10 particles as measured by NTA) including a negative control of LNCaP‐derived EVs (high PSMA expression) were labelled with anti‐PSMA antibodies and analysed by the AF4‐MALS‐FLD protocol. (F) The area under the curve for the EV peak was determined for LNCaP‐derived EVs. Different concentrations (2 × 10 10 , 4 × 10 10 and 6 × 10 10 particles as measured by NTA) including a negative control of (G) MCF‐7‐derived EVs (high EpCAM expression) or (I) SK‐BR‐3‐derived EVs (high HER2 expression) were labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively and analysed by the AF4‐MALS‐FLD protocol. The area under the curve for the EV peak (24–80 min) was determined for (H) MCF‐7‐ and (J) SK‐BR‐3‐derived EVs.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Derivative Assay, Multi-Angle Light Scattering, Negative Control, Expressing

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Detection of EVs in complex matrices . (A) Different volumes of cell culture supernatant (0, 20, 40 and 60 µL) collected from the MCF‐7 cells were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak in complex matrices (40–80 min) was determined. (B) Different amounts of LNCaP‐derived EVs were spiked in 100 µL of concentrated urine, diluted 1:1 in PBS to reduce viscosity, labelled with PE‐conjugated anti‐PSMA antibodies, and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak was determined. Different amounts of (C) MCF‐7‐ or (D) SK‐BR‐3‐derived EVs were spiked in 100 µL of blood plasma, diluted 1:1 in PBS to reduce viscosity, and labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies, respectively. Labelled EVs were analysed by AF4‐MALS‐FLD and the area under the curve for the EV peak was determined. Different amounts of SK‐BR‐3 EVs were also spiked in blood plasma and labelled with isotype control antibodies. (E) Different concentrations of soluble EpCAM (1, 5 and 10 ng/mL) and soluble HER2 (50, 100 and 150 ng/mL) were spiked in blood plasma, labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively, and analysed by AF4‐MALS‐FLD.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Cell Culture, Derivative Assay, Viscosity, Clinical Proteomics, Control

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Validation of the AF4‐MALS‐FLD workflow on patient samples . Urine samples of five prostate cancer patients were labelled for PSMA and analysed by the AF4‐MALS‐FLD workflow. Fractions 40–80 min were collected, concentrated and processed for mass spectrometry‐based proteomic analysis. (A) EV markers Syntenin‐1, Flotillin‐1, CD63, CD9, CD81, Flotillin‐2, Alix and TSG101 were analysed (missing sample indicated in grey). Z ‐score transformation of intensities were plotted. (B) Targeted mass spectrometry analysed the presence of PSMA (FOLH1) in patient samples. The z ‐score transformation of intensities was plotted with the AF4‐MALS‐FLD peak area. (C) Blood plasma samples of healthy controls ( n = 7) and HER2 amplified breast cancer patients ( n = 10) were labelled with PE‐conjugated anti‐HER2 antibodies. (D) Blood plasma samples of healthy controls ( n = 6) and breast cancer patients ( n = 8) were labelled with PE‐conjugated anti‐EpCAM antibodies. The area under the curve values were normalised for the mean value in the healthy control group.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Mass Spectrometry, Transformation Assay, Clinical Proteomics, Amplification, Control

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Regnase-1 downregulation promotes pancreatic cancer through myeloid-derived suppressor cell-mediated evasion of anticancer immunity.

doi: 10.1186/s13046-023-02831-w

Figure Lengend Snippet: Fig. 6 Suppression of CTLs by CD11b+ MDSCs is responsible for the acceleration of tumor progression by Regnase-1 downregulation. A-D Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1 KO murine pancreatic cancer cells. Representative macro images of pancreatic tumors (A). Relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (B) (N = 6 per group). Representative images of HE (C, left panel) and CD8a immunostaining (C, right panel) and the number of CD8-positive cells (C, right) (N = 6 per group). Dot plots of CD3+CD8+ cells evaluated by flow cytometry (D, left) and the proportion of CD8 + cells among CD45+ cells (D, right) (N = 3 per group). E–H Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1-KO murine pancreatic cancer cells with or without depletion of CD8+ cells upon anti-CD8a antibody or IgG treatment. Experimental schematic (E). Dot plots of CD3+ and CD8.+ cells in WT or Regnase-1-KO syngeneic tumors upon anti-CD8a antibody or IgG treatment evaluated by flow cytometry (F). Tumor weights (G) (N = 6 per group). The relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (H) (N = 6 per group). Student’s t test was used to evaluate differences between 2 groups. One-way ANOVA with Tukey’s post hoc test was used to compare differences among 4 groups. *P < 0.05, scale bars: 100 μm (insets)

Article Snippet: BE0061, a fully neutralizing mAb recognizing CD8a, and control IgG were obtained from Bioxcell.

Techniques: Immunostaining, Flow Cytometry

Journal: Biomolecules

Article Title: 1-(Arylsulfonyl-isoindol-2-yl)piperazines as 5-HT 6 R Antagonists: Mechanochemical Synthesis, In Vitro Pharmacological Properties and Glioprotective Activity

doi: 10.3390/biom13010012

Figure Lengend Snippet: ( A ): Functional dose-response curve of inhibition of cAMP production at 5-HT 6 R for selected compounds 3e , 3f , and 3g in 1321N1 cells. Data were obtained from three independent experiments run in triplicate. ( B ): Impact of compounds 3e , 3f , and 3g and SB-258585 on basal cAMP production in NG108-15 cells transiently expressing 5-HT 6 R. For each compound, six independent transfection experiments were performed, and data were measured in triplicate. Data are given as means ± SEM of the values.

Article Snippet: The ability of compounds 3e , 3f , and 3g to inhibit 5-CT-induced production of cAMP was assessed using 1321N1 cells expressing the human 5-HT 6 R (PerkinElmer) using previously described procedures [ , ].

Techniques: Functional Assay, Inhibition, Expressing, Transfection