Journal: Bioactive Materials

Article Title: Engineered extracellular vesicles for concurrent Anti-PDL1 immunotherapy and chemotherapy

doi: 10.1016/j.bioactmat.2021.07.012

Figure Lengend Snippet: Characterization of constructed cells. (a) The expression level of PD-L1, PD-1, and B2M as measured by flow cytometry in wild-type MDA-MB-231 and double-KO MDA-MB-231 (Red: blank; Blue: Isotype antibody; Yellow: anti-PD-L1, anti-PD-1, or anti-B2M antibody). (b) Fluorescent image of PD-L1, PD-1, and B2M in wild-type and double-KO MDA-MB-231 cells (scale bar is 10 μm). (c) The expression level of PD-L1 and havPD-1 as measured by flow cytometry and fluorescence microcopy in double-KO/havPD-1 knock-in MDA-MB-231 cells (Red: blank; Blue: Isotype antibody; Yellow: anti-PD-L1 or anti-PD-1 antibody; scale bare is 10 μm). (d) B2M, PD-L1, and havPD-1 proteins were extracted and identified from wild-type and constructed MDA-MB-231 cells. (e) Fluorescent image of constructed double-KO/havPD-1 knock-in/CD81-GFP fusion MDA-MB-231 cells (scale bar is 10 μm) and the expression level of CD81-GFP as measured by flow cytometry (Red: blank; Blue: MDA-MB-231 cells w/o CD81-GFP fusion protein; Yellow: MDA-MB-231 cells expressing CD81-GFP fusion protein). (f) Fluorescent image of constructed MDA-MB-231-tdTomato cells (scale bar is 10 μm).

Article Snippet: Anti-PD-1 magnetic beads immunoprecipitation kit (SinoBiological, MB108583-T36) was used to harvest havPD-1 as per the manufacturer's instructions.

Techniques: Construct, Expressing, Flow Cytometry, Fluorescence, Knock-In

Journal: Bioactive Materials

Article Title: Engineered extracellular vesicles for concurrent Anti-PDL1 immunotherapy and chemotherapy

doi: 10.1016/j.bioactmat.2021.07.012

Figure Lengend Snippet: Characterization of EVs. (a) Size distribution of havPD-1 EVs (left) and morphology characterization of havPD-1 EVs by electron microscopy (scale bar is 100 nm). (b) Western blot analysis of havPD-1 extracted from trypsin-treated havPD-1 EVs and free havPD-1. (c) TEM image of havPD-1 EVs stained with anti-PD-1 antibodies grafted gold nanoparticle (scale bar is 50 nm) and STEM image of surviving havPD-1 EVs stained with immunogold (red dots; scale bar is 100 nm). (d) Western blot analysis of CD47, CD55, CD59, CD9, TSG101, and GAPDH extracted from havPD-1 EVs (1) and havPD-1/GFP EVs (2). (e) Association and dissociation of havPD-1 or Atezolizumab to PD-L1 by SPR. (f) Size distribution of PD-L1 EVs (left) and morphology characterization of PD-L1 EVs by electron microscopy (scale bar is 100 nm). (g) Morphology of havPD-1 EVs, PD-L1 EVs, and their mixture (scale bar is 100 nm). (h) Size distribution of havPD-1/CD81-GFP EVs (left); morphology characterization of havPD-1/CD81-GFP EVs by electron microscopy (middle, scale bar is 100 nm); Fluorescence image of havPD-1/CD81-GFP EVs (right, scale bar is 1 μm). (i) Fluorescence images and quantified data of fluorescence intensity of each group (n = 15) showing PKH67-labeled PD-L1 EVs and havPD-1/GFP EVs taken up by MDA-MB-231-tdTomato cells in 30 min (scale bar is 10 μm).

Article Snippet: Anti-PD-1 magnetic beads immunoprecipitation kit (SinoBiological, MB108583-T36) was used to harvest havPD-1 as per the manufacturer's instructions.

Techniques: Electron Microscopy, Western Blot, Staining, Fluorescence, Labeling

Journal: Frontiers in Immunology

Article Title: Sustained Drug Release From Liposomes for the Remodeling of Systemic Immune Homeostasis and the Tumor Microenvironment

doi: 10.3389/fimmu.2022.829391

Figure Lengend Snippet: The tumor growth inhibition effects of L-ATRA combined with anti-PD-1 treatment. (A) Outline of the dose schedule; (B) Analysis of Tumor growth in different treatment groups; (C) . Individual tumor growth curves in different treatment groups.

Article Snippet: Therapeutic mouse anti-PD-1 were purchased from BIO X CELL™(USA), InVivoMAb Antibodies,anti-mouse PD-1(CD279,Clone: 29F.1A12).

Techniques: Inhibition

Journal: Frontiers in Oncology

Article Title: Low-density lipoprotein balances T cell metabolism and enhances response to anti-PD-1 blockade in a HCT116 spheroid model

doi: 10.3389/fonc.2023.1107484

Figure Lengend Snippet: The exhaustion markers PD-1 and LAG-3 were down-regulated in the presence of LDL high (here shown for the CD4 + subset after 96 h) and the costimulatory marker CD154 (CD40L) was upregulated in the CD4 + T cell subset. T cells were freshly isolated and stimulated with anti-CD3/CD28 beads, IL-2 and treated with different LDL dosages (LDL low = 50 µg/ml and LDL high = 200 µg/ml) versus medium control. PD-1: LDL high significantly reduced the fraction (A) of PD-1 + cells and the expression (B) of PD-1 on the CD4 + T cell subset after 96 h. Representative FACS plots and histograms are presented (A,B). LAG-3 : LDL high significantly reduced the fraction of LAG-3 + cells in the CD4 + T cell subset after 96 h. Representative FACS plots are presented (C) . CD154 : The fraction of CD154 (CD40L) positive cells (D) and the expression (E) of CD154 was significantly enhanced in the presence of LDL high in the CD4 + subset. Representative FACS plots and histograms are presented (D,E). Depending on normal or non-normal distribution, RM one-way ANOVA with Geisser-Greenhouse correction and Dunnett`s multiple comparison test or Friedman test with Dunn`s multiple comparison test was performed. Significance was indicated as p < 0.05 *, p < 0.01 ** referring to control. All data were corrected for multiple testing according to Benjamini and Hochberg.

Article Snippet: For some of the experiments as indicated, an anti-human PD-1 blocking antibody (InVivoMAb anti-human PD-1, Bio X Cell, BE0188) was added at a concentration of 10 µg/ml.

Techniques: Marker, Isolation, Expressing

Journal: Frontiers in Oncology

Article Title: Low-density lipoprotein balances T cell metabolism and enhances response to anti-PD-1 blockade in a HCT116 spheroid model

doi: 10.3389/fonc.2023.1107484

Figure Lengend Snippet: Referring to control, viable cells were significantly reduced in the HCT116 spheroid tumor model in the presence of LDL high in combination with an anti-PD-1 antibody. Tumor spheroids were generated for 4 days with HCT116 colon carcinoma cells. T cells were freshly isolated and stimulated with anti-CD3/CD28 beads, IL-2 and treated with anti-PD-1, LDL high or LDL high + anti-PD-1 versus medium control. After 48 h, T cells were added to the tumor spheroid and co-cultured for further 24 h. For Incucyte life cell imaging system, spheroids were washed, stained with a viability dye and fluorescence was then monitored under culture conditions for further 24 h – 48 h (cumulative T cell stimulation time 96 h – 120 h). (A) Spheroid co-culture experimental scheme. Created with BioRender.com. (B) Representative picture of spheroids after 24 h live cell imaging. Red = dead, green = viable. (C, D) Quantification of the green object total area (GOTA), which determines viable cells after 24 h (C) and 48 h live cell imaging (D) . Depending on normal or non-normal distribution, RM one-way ANOVA with Geisser-Greenhouse correction and Dunnett`s multiple comparison test or Friedman test with Dunn`s multiple comparison test was performed. Significance was indicated as p < 0.05 *, p < 0.01 ** referring to control. ns, not significant.

Article Snippet: For some of the experiments as indicated, an anti-human PD-1 blocking antibody (InVivoMAb anti-human PD-1, Bio X Cell, BE0188) was added at a concentration of 10 µg/ml.

Techniques: Generated, Isolation, Cell Culture, Imaging, Staining, Fluorescence, Cell Stimulation, Co-Culture Assay, Live Cell Imaging