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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 4. — Tumor cells re-challenge of cured mice and tumor cell vaccination.

a, Schematic of tumor re-challenge (upper) and tumor cell vaccination assay (bottom). In the tumor re-challenge study, CT26.WT-HER2-tumor bearing mice were completely cured with TS-L6 (20 mg/kg, once weekly × 2), followed by tumor re-challenge at days 60, 90 and 180 post-initial dosing. In the tumor vaccination assay, mice were vaccinated with 3 million tumor cells pre-treated with MF-6 or TS-L6, followed by tumor re-challengeat day 21.

b, When the tumor re-challenge at 2 months post-initial dosing was completed, the plasma antibody titers to CT26.WT-HER2 and CT26.WT cells were measured by a cell-based ELISA. The cured mice re-challenged with either CT26.WT-HER2 (upper) or CT26.WT (bottom) showed higher antibody titers to CT26.WT-HER2 or CT26.WT cells compared with naïve mice. Eight mice in each group were used, and the data are shown as mean and SD. The titer data were statistically analyzed after logarithm transformation.

c, Tumor re-challenge was conducted with mice cured by single-drug TS-6 (20 mg/kg, once weekly × 2) 2 months after initial dosing. All cured mice (8/8) rejected CT26.WT-HER2 tumor cells (black lines), whereas 3 of 8 of the cured mice rejected parental CT26.WT tumor cell re-challenge (blue lines). Neither the cured mice nor the naïve mice showed any tumor rejection when challenged with EMT6 (red lines). A log-rank test was used to compare the probability of survival, and the same method was for survival curve comparison.

d, All mice (8/8) cured by single-drug TS-L6 rejected CT26.WT-HER2 tumor cell challenge for months (black line) and 6 months (blue line) after initial dosing.

e, When the tumor re-challenged at 6 months post-initial dosing was completed, the plasma antibody titers to CT26.WT-HER2 and CT26.WT were measured with a cell-based ELISA. The cured mice re-challenged with CT26.WT-HER2 showed higher antibody titer to CT26.WT-HER2 or CT26.WT cells compared with naïve mice. Eight mice/group were used, and the data are shown as mean and SD. The data were statistically analyzed after logarithm transformation.

f, g, Mice vaccinated with CT26.WT-HER2 cells pre-treated with 10 nmol/L MF-6 for 24 h (blue line) or 100 nmol/L TS-L6 for 72 h (red line) exhibited a higher tumor rejection ratio and higher survival probability than mice vaccinated with necrotic cells (black line) when re-challenged with CT26.WT-HER2 (f) or parental CT26.WT (g).

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