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. 2023 Jun 14;15(1):2220466. doi: 10.1080/19420862.2023.2220466

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© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

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Figure 2. — Genomic DNA damage and DAMPs upregulation in tumor cells with drug treatment.

a, The drug release rate of TS-L6, TS-GGFG-DXd, and TS-vcMMAE was evaluated using cathepsin B and L at pH 4.5, 5.0, and 5.5. The cathepsin B and cathepsin L released 45% and 25% MF-6 from TS-L6 at the optimal pH condition after 96 h. The GGFG linker was more sensitive to cathepsin L, which released approximately all the DXd after 72 h; cathepsin B barely catalyzed DXd release. The traditional VC linker was more sensitive to both cathepsin B and L compared with VA and GGFG linker; after only 0.5 h, approximately all the MMAE was released. The release rates were TS-L6<TS-GGFG-DXd<TS-vcMMAE. Three independent tests were performed, and the data are shown as mean and SD.

b, The drug release of TS-L6 and TS-GGFG-DXd in tumor cells was evaluated on NCI-N87. After incubation for different times, the released drugs in cells were isolated for quantitative analysis. The released MF-6 in tumor cells was lower compared with released DXd at all the times (left), and the ratio of released MF-6 to total MF-6 carried by internalized TS-L6 was also lower compared with DXd (right). Three independent tests were performed, and the data are shown as mean and SD.

c, Genomic DNA of NCI-N87, indicated by the Tert gene, was released into the cytoplasm after MF-6 and TS-L6 treatment. When treated with 2 nmol/L MF-6 for 3 days, the amount of genomic DNA of NCI-N87 was significantly increased compared with DMSO (2.07 ± 0.48 vs. 1.27 ± 0.18, P<0.05). Similarly, when treated with 50 nmol/L TS-L6 for 5 days, the amount of genomic DNA was significantly increased compared with 50 nmol/L TS (4.71 ± 1.38 vs. 1.24 ± 0.42, P<0.01). Three independent tests were performed, and the data are shown as mean and SD.

d, Phosphorylated H2A.X (γ-H2A.X) level of NCI-N87 treated with MF-6 and TS-L6, which was a biomarker of genomic DNA damage, was assessed by immunofluorescence. The γ-H2A.X (green, stained with phosphorylated H2A.X antibody) was located in the nucleus (blue, stained with DAPI) and its nuclear abundance increased after MF-6 and TS-L6 treatment, thereby indicating genomic DNA damage.

e, When NCI-N87 and CT26.WT-HER2 were treated with 2 nmol/L MF-6 for 3 days or 50 nmol/L TS-L6 for 5 days, the cell surface DAMPs calreticulin, HSP70, and HSP90 significantly increased, compared with DMSO or 50 nmol/L TS treatment. For NCI-N87 cells, MF-6 treatment significantly increased calreticulin (1.74 ± 0.03 vs. 0.94 ± 0.03 folds, P<0.001), HSP70 (1.34 ± 0.06 vs. 1.08 ± 0.02 folds, P<0.001), and HSP90 (1.86 ± 0.05 vs. 0.96 ± 0.05 folds, P<0.001) on cell surface compared with DMSO. Similarly, compared with the 50 nmol/L TS treatment, 50 nmol/L TS-L6 treatment led to increase in calreticulin (1.78 ± 0.11 vs. 1.21 ± 0.04 folds, P<0.001), HSP70 (1.27 ± 0.09 vs. 1.01 ± 0.08 folds, P<0.01), and HSP90 (1.97 ± 0.13 vs. 1.01 ± 0.05 folds, P<0.001). Mouse cell line CT26.WT-HER2 also exhibited upregulated DAMPs after 2 nmol/L MF-6 or 50 nmol/L TS-L6 treatment. Six independent tests were performed, and the data are shown as mean and SD.

f, When NCI-N87 and CT26.WT-HER2 were treated with 2 nmol/L MF-6 for 3 days or 50 nmol/L TS-L6 for 5 days, the MHC-I, MHC-II, and PD-L1 on the tumor cell surface significantly increased, compared with DMSO or 50 nmol/L TS treatment. For NCI-N87 cells, 2 nmol/L MF-6 treatment significantly increased MHC-I (1.77 ± 0.04 vs. 0.97 ± 0.07 folds, P<0.001), MHC-II (1.91 ± 0.28 vs. 0.98 ± 0.07 folds, P<0.01) and PD-L1 (2.46 ± 0.09 vs. 0.99 ± 0.03 folds, P<0.001) on cell surface compared with DMSO. Similarly, compared with the 50 nmol/L TS treatment, 50 nmol/L TS-L6 treatment led to increase in MHC-I (1.53 ± 0.07 vs. 1.00 ± 0.03 folds, P<0.001), MHC-II (2.35 ± 0.50 vs. 0.85 ± 0.14 folds, P<0.01) and PD-L1 (1.47 ± 0.16 vs. 0.96 ± 0.05 folds, P<0.001). Mouse tumor cell CT26.WT-HRR2 also exhibited upregulated MHC-I, MHC-II, and PD-L1 after MF-6 or TS-L6 treatment. Six independent tests were performed, and the data are shown as mean and SD.

ns, *, ** and *** represent no significance, P<0.05, 0.01, and 0.001, respectively.