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. 2024 Jul 23;15:6211. doi: 10.1038/s41467-024-50474-y

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — a Scheme of the construction of Hu Tri-NAb through gentle mixing of Hu APCN@NA and Hu-αPDL1, Hu-α4-1BB, and Hu-αTIGIT. Similar to Fig. 2i., Hu APCN@NA was prepared by coupling Hu αFc on the surface of APCN. b Size distribution of Hu APCN@NAs and Hu Tri-NAb determined using DLS. c An experimental scheme illustrating the study of the cytotoxicity of human primary CD56⁺ NK and CD8⁺ T cells from human peripheral blood mononuclear cells (PBMCs) toward colon cancer organoids from patient-derived colon cancer tissues after an incubation with 1) control, 2) Hu Tri-mAbs (Hu-αPDL1 & Hu-α4-1BB & Hu-αTIGIT), 3) NP_{αPDL1+α4-1BB}, 4) NP_{αPDL1+αTIGIT}, 5) NP_{α4-1BB+αTIGIT}, or 6) Hu Tri-NAb. The organoids were first labeled with CFSE, and all the cells were stained with propidium iodide (PI). The proportion of CFSE⁺ PI⁺ cells was detected using flow cytometry. d Bright-field image of colon cancer organoids. e Fluorescence image of CFSE-labeled colon cancer organoids. f Representative scatter plots of flow cytometry data and corresponding quantification results (g) showing the number of CFSE⁺ PI⁺ cells as a percentage of the total cells. Data are presented as means ± s.d. (n = 4 biologically independent samples). h Experimental scheme of the subcutaneous HT-29 human colon xenograft tumor model in female NCG-hIL15 mice. i Average tumor growth kinetics in mice treated with different formulations. Data are presented as means ± s.d. (n = 4 mice per group). j Tumor images collected from treatment endpoints. k Tumor weights after different treatments. Data are presented as means ± s.d. (n = 4 mice per group). The levels of human IFN-γ (l), human granzyme B (m), and human perforin (n) in treated HT-29 tumors were examined using ELISAs. Data are presented as the means ± s.d. (n = 4 biologically independent samples). o Representative fluorescence images of tumor lesions after staining with DAPI (blue), anti-human CD56 (FITC, green), and anti-human CD3 antibodies (Cy3, red). Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. Figures 7c and h were created with BioRender.com under a CC-BY-NC-ND license. Source data are provided as a Source Data file.