Journal: Science Advances
Article Title: Recruitment of CD103+ dendritic cells via tumor-targeted chemokine delivery enhances efficacy of checkpoint inhibitor immunotherapy
Figure Lengend Snippet: CBD-CCL4 exhibits high affinity to collagen and accumulates in tumor following intravenous injection. ( A ) WT CCL4 and CBD-CCL4 were analyzed by SDS-PAGE followed by Coomassie blue staining. ( B and C ) Affinity of CBD-CCL4 against (B) collagen I and (C) collagen III was measured by SPR. SPR chips were functionalized with collagen I [~500 resonance units (RU)] and collagen III (~700 RU), and CBD-CCL4 was flowed over the chips at indicated concentrations. Curves represent the obtained specific responses (in resonance units) to CBD-CCL4. Experimental curves were fitted with 1:1 Langmuir fit model. Binding kinetics values [dissociation constants ( K d ) and rate constants ( k on and k off )] determined from the fitted curves are shown. ( D and E ) Binding of (D) WT CCL4 or (E) CBD-CCL4 to human melanoma cryosections as determined by immunofluorescence microscopy. Scale bars, 100 μM. DAPI, 4′,6-diamidino-2-phenylindole. ( F ) GPCR activation assay comparing signaling of WT CCL4 and CBD-CCL4 in THP1 monocytes. Median effective concentration (EC 50 ) values were calculated using a nonlinear dose-response curve fit model. Each point represents mean ± SEM, n = 3. ( G ) Blood plasma pharmacokinetics was analyzed using DyLight 800–labeled WT CCL4 or CBD-CCL4 in B16F10 melanoma. Four days after tumor inoculation, mice were administered 25 μg of WT CCL4 or the molar equivalent of CBD-CCL4 (25 μg of CCL4 basis or 93 μg of CBD-CCL4) via intravenous injection. Blood was collected at the indicated time points, and plasma was separated and analyzed for CCL4 concentration. Each point represents mean ± SEM, n = 4. ( H ) Biodistribution was analyzed using DyLight 647–labeled WT CCL4 or CBD-CCL4 in EMT6 breast cancer. When the tumor volume reached 500 mm 3 , 25 μg of WT CCL4 or the molar equivalent of CBD-CCL4 (25 μg of CCL4 basis or 93 μg of CBD-CCL4) was given via intravenous injection. Fluorescence intensity in each tumor was measured using an in vivo imaging system (IVIS), converted to percent injected dose using a known standard series, and normalized to the weight of the tumor. Each bar represents mean ± SEM, n = 3. ** P
Article Snippet: CBD-CCL4 binding to human and murine melanoma cryosections Human melanoma cryosections were obtained from OriGene Technologies.
Techniques: Injection, SDS Page, Staining, SPR Assay, Binding Assay, Immunofluorescence, Microscopy, Activation Assay, Concentration Assay, Labeling, Mouse Assay, Fluorescence, In Vivo Imaging