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. 2024 Apr 2;73(5):92. doi: 10.1007/s00262-024-03692-8

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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. 3 — TLC388 synergistically enhanced RT-induced cancer immunogenicity. (A) The schematic diagram of immature DC cocultured with TLC388-treatment HT29 cell and analyzed DC maturation marker by flow cytometry. (B) The TLC388 (0.5 µM)-treated HT29 cells were cocultured with THP1-iDC cells, which were differentiated into immature DC (iDC) by IL-4 (1500 IU/ml) and GM-CSF (1500 IU/ml) for 7 days, for 24 h. The DC marker (CD86) was evaluated by flow cytometry. **p < 0.01. One-way ANOVA t-test. (C) HT29 cells were treated with TLC388 (0.5 µM) for 24 h, and then washout the drugs to coculture with THP1-iDC for 24 h. The mRNA level of DC markers (CD86 and CD80) was determined by qRT-PCR(n = 3). *p < 0.05 and **p < 0.01. One-Way ANOVA t-test. (D) HT29 and CT26 cells were irradiated for 5 Gy and treated with TLC388 (0.5 and 1 µM) for 24 h. The cell lysate was harvested for western blot analysis. (E) HT29 cells were irradiated with 5 Gy and treated with TLC388 (0.5 and 1 µM) for 24 h. The level of IFNβ1, CXCL10, IL12a, and TNFα mRNA was analyzed by qRT-PCR (n = 3). *p < 0.05 and **p < 0.01. One-way ANOVA t-test. (F) CT26 cells were irradiated with 5 Gy and treated with TLC388 (0.5 and 1 µM) for 24 h. The level of IFNβ1, CXCL10, IL12a, and TNFα mRNA was analyzed by qRT-PCR (n = 3). *p < 0.05 and **p < 0.01. One-way ANOVA t-test. (G) Tumor growth of CT26-driven colon carcinoma established in BALC/c mice (n = 6 per group) that were treated with RT (5 Gy for 2 fractions) and RT/TLC (TLC:2.5 mg/kg). Tumor growth is reported as the mean tumor volume ± SD. *p < 0.05 and **p < 0.01. CR: complete response. Two-way ANOVA t-test. (H) The tumor-infiltrating CD11c⁺ DC and GzmB⁺ immune cells within resected tumors were analyzed by immunofluorescent staining (n = 3). (I) The quantification of tumor-infiltrating CD11c⁺ dendritic cells within resected tumors (n = 3). *p < 0.05 and **p < 0.01. One-way ANOVA t-test. (J) The quantification of tumor-infiltrating GzmB⁺ T cells within resected tumors (n = 3). *p < 0.05 and **p < 0.01. One-way ANOVA t-test