permeabilization buffer  (R&D Systems)

Journal: Science Advances

Article Title: Tumor-specific delivery of clickable inhibitor for PD-L1 degradation and mitigating resistance of radioimmunotherapy

doi: 10.1126/sciadv.adq3940

Figure Lengend Snippet: ( A ) Synthetic routes of the clickable PD-L1 inhibitors. ( B and C ) Flow cytometry examination of PD-L1 abundance and quantification of inhibition efficiency on the membrane surface of (B) wild-type 4T1 tumor cells and (C) wild-type B16-F10 tumor cells [median fluorescence intensity (MFI)]. The tumor cells were first incubated with Ac 4 ManAz (25 μM) for 3 days to label PD-L1 with azide groups and then treated with the clickable PD-L1 inhibitors for 24 hour. Last, the PD-L1 abundance on the membrane surface was examined by flow cytometry. ( D ) Schematic illustration of the positive correlation between the OEG linker length of the clickable PD-L1 inhibitors and their PD-L1 degradation efficacy. ( E and F ) Flow cytometry–determined PD-L1 abundance on the surface of (E) wild-type 4T1 tumor cells and (F) wild-type B16-F10 tumor cells without or without azide labeling. ( G ) A proposed mechanism for clickable PD-L1 inhibitor–mediated PD-L1 degradation via bioorthogonal click chemistry and metabolic glycan engineering, which is superior over the conventional inhibitors via physical binding. ( H ) Flow cytometry determined the membrane surface PD-L1 degradation profile of the clickable PD-L1 inhibitor in various human and murine tumor cell lines. The data are presented as means ± SD.

Article Snippet: To stain intracellular proteins, the cell suspension was fixed and permeabilized with the commercial buffer Flow Cytometry Permeabilization/Wash Buffer I (R&D Systems), followed by intracellular staining with anti–IFN-γ–FITC.

Techniques: Flow Cytometry, Inhibition, Membrane, Fluorescence, Incubation, Labeling, Binding Assay

Journal: Science Advances

Article Title: Tumor-specific delivery of clickable inhibitor for PD-L1 degradation and mitigating resistance of radioimmunotherapy

doi: 10.1126/sciadv.adq3940

Figure Lengend Snippet: ( A ) Schematic illustration of IFN-γ–induced PD-L1 up-regulation on the surface of tumor cells in vitro. ( B to E ) Flow cytometry and Western blot examination of clickable PD-L1 inhibitor–mediated PD-L1 degradation on the surface of tumor cell membrane in vitro. [(B) and (C)] Flow cytometry detection of PD-L1 abundance on the membrane surface of IFN-γ–pretreated 4T1 (B) and B16-F10 (C) tumor cells. [(D) and (E)] Western blot analysis of PD-L1 abundance on the membrane surface of IFN-γ–pretreated 4T1 (D) and B16-F10 (E) tumor cells. (F) Representative CLSM images of PD-L1 abundance on the membrane surface of 4T1 tumor cells (scale bar = 20 μm). Na + ,K + -ATPase, Na + - and K + -dependent adenosine triphosphatase. ( G to J ) Flow cytometry–determined PD-L1 abundance on the membrane surface of IFN-γ–pretreated 4T1 (G) and B16-F10 (H) tumor cells after treatment with 10 μM BMS-1 or D5B. Western blot analysis and semi-quantification of PD-L1 expression on the membrane surface of IFN-γ–pretreated 4T1 (I) and B16-F10 (J) tumor cells with or without azide labeling. ( K ) Representative flow cytometry plots and quantification of IFN-γ + CD8 + T cells. 1#, CD8 + T cells incubated with BMS-1–treated tumor cells; 2#, CD8 + T cells incubated with D5B-treated tumor cells; 3#, CD8 + T cells incubated with PBS. ( L ) Mechanism illustration for PD-L1 degradation increased proliferation of CD8 + T lymphocytes. The data were presented as the means ± SD. P values were determined by two-way repeated-measures analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test [(D) and (E)], unpaired Student’s t test [(I) and (J)], or one-way ANOVA with Tukey’s multiple comparisons test (L). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: To stain intracellular proteins, the cell suspension was fixed and permeabilized with the commercial buffer Flow Cytometry Permeabilization/Wash Buffer I (R&D Systems), followed by intracellular staining with anti–IFN-γ–FITC.

Techniques: In Vitro, Flow Cytometry, Western Blot, Membrane, Expressing, Labeling, Incubation

Journal: Science Advances

Article Title: Tumor-specific delivery of clickable inhibitor for PD-L1 degradation and mitigating resistance of radioimmunotherapy

doi: 10.1126/sciadv.adq3940

Figure Lengend Snippet: ( A ) Acid-responsive mechanism illustration of the pH e -activatable PCP@D5B, pH i -activatable PDP@D5B, and pH-inactivated PBP@D5B nanoparticles for acid-triggered nanoparticle dissociation and activation of NIRF/MRI signals. ( B ) Dynamic light scattering (DLS)– and transmission electron microscopy (TEM)–determined particle size distribution and morphology change of the PCP@D5B, PDP@D5B, and PBP@D5B nanoparticles at pH 7.4 and 6.5, respectively (scale bars, 100 nm). ( C ) Representative CLSM images of cell membrane [wheat germ agglutinin (WGA), green] and colocalization with PPa-labeled nanoparticles (red) at the predesignated time points. 4T1 tumor cells were incubated with PCP@D5B, PDP@D5B, or PBP@D5B nanoparticles for 5 min under different pH conditions (scale bars, 20 μm). ( D ) Schematic illustration for PCP@D5B-performed PD-L1 degradation on the surface of tumor cell membrane by specifically releasing D5B payload at the extracellular acidic microenvironment in vitro. ( E ) Flow cytometry analysis of PD-L1 abundance on the surface of 4T1 tumor cell membrane; inset number represents the MFI values. ( F ) The mechanism of pH-activated NIRF and MRI signals of PCPGd@D5B nanoparticles. ( G ) Representative T 1 maps of PCPGd@D5B nanoparticles determined at varied pH values. ( H ) The longitudinal relaxation rate ( r 1 ) versus Gd 3+ concentration determined at different pH values. ( I ) MRI of 4T1 tumor-bearing mice in vivo. The mice were intravenously injected with PBS or PCPGd@D5B nanoparticles at a Gd 3+ dose of 1.5 mg/kg and then imaged at the predetermined intervals (white circles represent the tumors). The data are presented as the means ± SD. h, hours.

Article Snippet: To stain intracellular proteins, the cell suspension was fixed and permeabilized with the commercial buffer Flow Cytometry Permeabilization/Wash Buffer I (R&D Systems), followed by intracellular staining with anti–IFN-γ–FITC.

Techniques: Activation Assay, Transmission Assay, Electron Microscopy, Membrane, Labeling, Incubation, In Vitro, Flow Cytometry, Concentration Assay, In Vivo, Injection

Journal: Science Advances

Article Title: Tumor-specific delivery of clickable inhibitor for PD-L1 degradation and mitigating resistance of radioimmunotherapy

doi: 10.1126/sciadv.adq3940

Figure Lengend Snippet: ( A ) Schematic illustration of pH-triggered extracellular delivery of D5B for PD-L1 degradation. ( B ) Representative IVIS fluorescence images of 4T1 tumor-bearing BALB/c mice in vivo. ( C ) Semiquantitative of PPa fluorescence intensity from (B) ( n = 3 mice). ( D ) High-performance liquid chromatography (HPLC)–determined pharmacokinetics of D5B-loaded PCP@D5B, PDP@D5B, and PBP@D5B nanoparticles or free D5B ( n = 3 mice). ( E ) HPLC-determined D5B distribution in the tumor mass after intravenous injection ( n = 3 mice). ( F ) Experimental schedule for antitumor study in vivo. it, intratumoral; iv, intravenous; sc, subcutaneous. ( G and H ) Averaged tumor growth curves (G), and (H) animal survival curves of 4T1 tumor-bearing mice (n = 6 mice). ( I and J ) Immunohistochemical (IHC) (I) and flow cytometry (J) examination of PD-L1 abundance 3 days after treatment ( n = 3 mice; scale bars, 50 μm). ( K ) Flow cytometry examination of tumor-infiltrating CD8 + and CD4 + T cells (gated on CD3 + CD45 + ) ( n = 5 mice). ( L ) Flow cytometry examination of tumor-infiltrating IFN-γ + CD8 + T cells (n = 5 mice). ( M and N ) Tumor mass normalized number of tumor-infiltrating CD8 + (M) and IFN-γ + CD8 + T cells (N) ( n = 5 mice). ( O ) Enzyme-linked immunosorbent assay (ELISA) analysis of intratumoral IFN-γ cytokine secretion at 1, 3, and 7 days after treatment ( n = 3 mice). ( P ) IHC examination of PD-L1 abundance in the normal tissue 3 days after the treatment. ( Q ) Schematic description for tumor-specific delivery of D5B and PD-L1 inhibition with the pH e -activatable nanoparticles. All data are presented as the means ± SD. P values were determined by one-way ANOVA with Tukey’s post hoc test [(J) to (N)], repeated-measures two-way ANOVA with Tukey’s multiple comparisons test [(E), (G), and (O)], log-rank test (H), or unpaired Student’s t test (P). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. n.s., not significant.

Article Snippet: To stain intracellular proteins, the cell suspension was fixed and permeabilized with the commercial buffer Flow Cytometry Permeabilization/Wash Buffer I (R&D Systems), followed by intracellular staining with anti–IFN-γ–FITC.

Techniques: Fluorescence, In Vivo, High Performance Liquid Chromatography, Injection, Immunohistochemical staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Science Advances

Article Title: Tumor-specific delivery of clickable inhibitor for PD-L1 degradation and mitigating resistance of radioimmunotherapy

doi: 10.1126/sciadv.adq3940

Figure Lengend Snippet: ( A ) Treatment schedule of in 4T1 tumor model in vivo. ( B ) Individual 4T1 tumor growth curves [complete regression (CR)] ( n = 6 mice). ( C ) Survival rates of 4T1 tumor-bearing mice. ( D ) Flow cytometry analysis of CD86 + CD80 + DCs ( n = 3 mice). ( E ) Flow cytometry examination of tumor-infiltrating CD8 + and CD4 + T cells, and ( F ) IFN-γ + CD8 + T cells ( n = 5 mice). ( G to I ) Absolute numbers of tumor-infiltrating CD3 + (G), CD8 + (H), and IFN-γ + CD8 + (I) T cells after the indicated treatments ( n = 5 mice). ( J ) M2/M1 ratio after treatment ( n = 5 mice). ( K ) Flow cytometry–determined PD-L1 abundance on the surface of tumor cells membrane ( n = 5 mice). ( L ) Treatment schedule of 4T1 abscopal tumor model (T1 and T2 represents the primary and abscopal tumors, respectively). ( M ) Averaged tumor growth curves ( n = 6 mice), and ( N ) Survival rates of the mice ( n = 6 mice). ( O ) Flow cytometry–determined tumor-infiltrating CD8 + T cells. ( P ) Flow cytometry analysis of T EM cells (CD62L − CD44 + ) in the spleens of 4T1 tumor-bearing mice ( n = 5 mice). ( Q ) Hematoxylin and eosin staining and quantification of metastatic tumor lesions in the lung ( n = 6 mice; scale bars, 2.5 mm). ( R ) Mechanism illustration for combinatory therapy–elicited antitumor immunity and immunological memory to suppress abscopal tumor and lung metastases. The data are presented as the means ± SD. P values were determined by repeated-measures two-way ANOVA with Tukey’s multiple comparisons test [(B) and (M)], log-rank test [(C) and (N)], or one-way ANOVA with Tukey’s post hoc test [(D) to (K) and (O) to (Q)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: To stain intracellular proteins, the cell suspension was fixed and permeabilized with the commercial buffer Flow Cytometry Permeabilization/Wash Buffer I (R&D Systems), followed by intracellular staining with anti–IFN-γ–FITC.

Techniques: In Vivo, Flow Cytometry, Membrane, Staining

Journal: Science Advances

Article Title: Tumor-specific delivery of clickable inhibitor for PD-L1 degradation and mitigating resistance of radioimmunotherapy

doi: 10.1126/sciadv.adq3940

Figure Lengend Snippet: ( A ) Treatment schedule for the antitumor study in B16-F10 tumor-bearing mice in vivo. ( B ) The averaged and individual B16-F10 tumor growth curves, and ( C ) survival curves of B16-F10 tumor-bearing mice monitored during the therapy period (CR represents the fractions of complete tumor regression at the end of antitumor study, n = 6 or 7 mice). ( D and E ) Representative flow cytometry plots (D), and quantification data of CD86 + CD80 + DCs (E) ( n = 3 mice). ( F and G ) Flow cytometry–determined fractions of (F) M2-phenotype (CD11b + CD206 + ) and (G) M1-phenotype (CD11b + CD80 + ) TAMs ( n = 5 mice). ( H ) The M2/M1 ratio of TAMs. ( I and J ) The MFIs of PD-L1 + TAMs (CD11b + CD80 + ) (I), and PD-L1 + CD45 − tumor cells (J) after treatment. ( K ) Immunofluorescence staining and semi-quantitation of PD-L1 + TAMs in the tumor sections (scale bars, 40 μm). ( L and M ) Representative flow cytometry plots (L) and quantification (M) of tumor-infiltrating CD8 + and CD4 + T cells (gated on CD45 + CD3 + ) ( n = 5 mice). ( N ) Immunofluorescence staining and semi-quantitation of PD-L1 + TAMs, and CD8 + T cells in the tumor sections (scale bars, 40 μm). ( O ) Schematic illustration of the clickable PD-L1 inhibitor mitigated the acquired immune evasion. RT induces ITM by up-regulating PD-L1 and recruiting M2-type TAMs, which was reversed with the clickable PD-L1 inhibitor through degrading PD-L1 on the surface of tumor cell membrane and repolarizing M2-type TAMs to M1 type. The data are presented as the means ± SD. P values were determined by repeated-measures two-way ANOVA with Tukey’s multiple comparisons test (B), log-rank test (C), or one-way ANOVA with Tukey’s post hoc test [(E) to (J) and (M)], * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: To stain intracellular proteins, the cell suspension was fixed and permeabilized with the commercial buffer Flow Cytometry Permeabilization/Wash Buffer I (R&D Systems), followed by intracellular staining with anti–IFN-γ–FITC.

Techniques: In Vivo, Flow Cytometry, Immunofluorescence, Staining, Quantitation Assay, Membrane