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. 2023 Mar 1;8:97. doi: 10.1038/s41392-023-01337-3

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© The Author(s) 2023

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — PMN-MDSCs were significantly augmented in mice bearing CCL20-overexpressing breast cancer cell orthotopic allograft tumors. a pSIN-/CCL20-overexpressing 4T1 cell lines were established, and overexpression efficiency of CCL20 was confirmed by qRT-PCR. Bar graph was presented as the mean of three biological independent experiments (mean ± SEM). Balb/c mice were orthotopically transplanted with pSIN-/CCL20-overexpressing 4T1 cells (5 × 10⁴) at the fourth mammary fat pads (n = 5 for each group). b The level of CCL20 in the blood serum of Balb/c mice was measured by ELISA and shown in bar graph as mean ± SEM. c Tumor size was monitored every 3 days, and tumor volume was calculated. d, e Tumor weight (d) and tumor image (e) was shown after the mice were sacrificed. f The percentage of MDSC (CD45⁺CD11b⁺Gr-1⁺) in pSIN-/CCL20-overexpressing 4T1 allograft tumors was analyzed by flow cytometry and shown in bar graph as mean ± SEM. g, h The percentage of CD3⁺CD8⁺ T cells (g) and the percentage of CD3⁺CD8⁺IFNγ⁺ T cells (h) from CD45⁺ cells in pSIN-/CCL20-overexpressing 4T1 cell allograft tumors were analyzed by flow cytometry and shown in bar graph as mean ± SEM. i–k The gating strategies for the analysis of PMN-MDSCs (CD45⁺CD11b⁺Ly6C^lowLy6G⁺) and M-MDSCs (CD45⁺CD11b⁺Ly6C^highLy6G⁻) by flow cytometry were shown (i). The percentage of PMN-MDSC (j) and M-MDSC (k) in tumor, spleen, blood, and BM of Balb/c mice bearing pSIN-/CCL20-overexpressing 4T1 cell allograft tumors were shown in bar graph as mean ± SEM. l The percentage of CD11b-positive cells and Ly6G-positive cells in Balb/c mice bearing pSIN-/CCL20-overexpressing 4T1 cell allograft tumors were analyzed by IF staining. Scale bar, 50 μm. m CD33 and CD15 expression in CCL20^high and CCL20^low tumor tissues from breast cancer patients were analyzed by IHC staining. Representative images and the graph of H Scores (mean ± SEM) were shown. Scale bar, 100 μm. ns, no significance; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001