Journal: Nature Communications

Article Title: Capturing nascent extracellular vesicles by metabolic glycan labeling-assisted microfluidics

doi: 10.1038/s41467-023-42248-9

Figure Lengend Snippet: Tumor-bearing mice were simultaneously treated with PD-L1 antibody and Ac 4 ManNAz (tetraacetylated N-Azidoacetyl-mannosamine), an unnatural sugar for metabolic labeling of EVs with azido groups. Subsequently, the azido-labeled EVs were tagged with biotin groups by click chemistry and captured by streptavidin (SA)-modified herringbone microfluidic chip.

Article Snippet: Mouse anti-human CD63 antibody (20 μg/mL, Cat#556019) was purchased from BD Pharmingen (USA), mouse anti-PD-L1 antibody (20 μg/mL, Cat#NBP1-43262) (detection antibody) was obtained from Novus Biologicals (USA), goat anti-mouse IgG H&L (beta-galactosidase) (120 μg/mL, Cat#ab136775) and rabbit anti-rat IgG H&L (beta-galactosidase) (120 μg/mL, Cat#ab136716) were purchased from Abcam (USA).

Techniques: Labeling, Modification

Journal: Nature Communications

Article Title: Capturing nascent extracellular vesicles by metabolic glycan labeling-assisted microfluidics

doi: 10.1038/s41467-023-42248-9

Figure Lengend Snippet: a Schematic schedule of the in vivo MGL in a 4T1 tumor-bearing mouse model. b Protocol diagram of the collection process for tissue-derived EVs. c Schematic of the detection of biotin-linked MGL CD63 + EVs and the detected distribution in plasma and different tissue samples. The fluorescence detection was on CD63 + EVs from plasma, tumor tissue and other major organs of 4T1 tumor-bearing mice with MGL, as well as CD63 + EVs from plasma and tumor tissue of 4T1 tumor-bearing mice without MGL. ΔFL = FL – FL 0 , where FL 0 and FL are the fluorescence intensity detected by Melac-Chip before and after the addition of sample. n = 5 biologically independent experiments. Data shown as mean ± SD. d Schematic of the detection of biotin-linked MGL PD-L1 + EVs. e The detected fluorescence intensity of PD-L1 + EVs from plasma samples of 4T1 tumor-bearing mice ( n = 3 biologically independent experiments) with different injection manners of Ac 4 ManNAz. I. t. refers to intratumoral injection and i. p. refers to intraperitoneal injection. Data shown as mean ± SD. f The detected fluorescence intensity of PD-L1 + EVs from plasma samples of normal mice and 4T1 tumor-bearing mice with Ac 4 ManNAz treatment ( n = 5 biologically independent experiments). Data are presented as mean values ± SD. g Normalized PD-L1 fluorescence intensity of plasma samples ( n = 3 biologically independent experiments) from 4T1 tumor-bearing mice at 0, 1, 2, 3 and 4 d injection of Ac 4 ManNAz (once/day). “3 d + and 7 d -” indicates a stop for 7 days after 3 consecutive days of injection. Data shown as mean ± SD. Statistical significance was determined using Tukey’s Method with One-Way ANOVA. P = 0.1063 (0 d vs 1 d), and P = 0.0005 (0 d vs 2 d). *** P < 0.001, and n.s. indicates non-significance ( P > 0.05). Source data are provided as a Source Data file.

Article Snippet: Mouse anti-human CD63 antibody (20 μg/mL, Cat#556019) was purchased from BD Pharmingen (USA), mouse anti-PD-L1 antibody (20 μg/mL, Cat#NBP1-43262) (detection antibody) was obtained from Novus Biologicals (USA), goat anti-mouse IgG H&L (beta-galactosidase) (120 μg/mL, Cat#ab136775) and rabbit anti-rat IgG H&L (beta-galactosidase) (120 μg/mL, Cat#ab136716) were purchased from Abcam (USA).

Techniques: In Vivo, Derivative Assay, Clinical Proteomics, Fluorescence, Injection

Journal: Nature Communications

Article Title: Capturing nascent extracellular vesicles by metabolic glycan labeling-assisted microfluidics

doi: 10.1038/s41467-023-42248-9

Figure Lengend Snippet: a Schematic of the tumor implantation, PD-L1 antibody immunotherapy, metabolic glycan labeling and sample collection on a 4T1-bearing mouse model. b Tumor growth curves of 4T1-bearing mice with/without PD-L1 antibody treatment. Data shown as mean ± SD. c Representative images of tumor tissue by HE and immunofluorescent staining. d – f Schematics of the detection of nascent CD63 + EVs ( d ), nascent PD-L1 + EVs ( e ), and total PD-L1 + CD63 + EVs ( f ) as well as the detected concentrations at different time points. Data shown as mean ± SD. Statistical significance was determined by a two-tailed unpaired t- test. For nascent PD-L1 + EVs, treated vs untreatment group: P = 0.0001 (17 d), P = 5.2209 × 10 −6 (24 days) ( e ). For total PD-L1 + CD63 + EVs, treated vs untreatment group : P = 0.0035 (17 d), P = 2.0980 × 10 −6 (24 days) ( f ). ** P < 0.01, *** P < 0.001, **** P < 0.0001. g Pearson correlation of the nascent PD-L1 + EVs (orange) and total PD-L1 + CD63 + EVs (black) to the tumor volume in 4T1-bearing mice with /without anti-PD-L1 treatment. n = 5 biologically independent experiments for the untreatment group, n = 6 biologically independent experiments for the treatment group. Correlations were determined by Pearson’s r coefficient. A two-tailed value of P < 0.05 was considered statistically significant. Source data are provided as a Source Data file.

Article Snippet: Mouse anti-human CD63 antibody (20 μg/mL, Cat#556019) was purchased from BD Pharmingen (USA), mouse anti-PD-L1 antibody (20 μg/mL, Cat#NBP1-43262) (detection antibody) was obtained from Novus Biologicals (USA), goat anti-mouse IgG H&L (beta-galactosidase) (120 μg/mL, Cat#ab136775) and rabbit anti-rat IgG H&L (beta-galactosidase) (120 μg/mL, Cat#ab136716) were purchased from Abcam (USA).

Techniques: Tumor Implantation, Glycoproteomics, Labeling, Staining, Two Tailed Test