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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. 3 — CCL20-modulated PMN-MDSCs enhanced the stemness of breast cancer cells. Balb/c mice or C57BL/6N mice were orthotopically transplanted with pSIN-/CCL20-overexpressing 4T1 or Py8119 cells at the fourth mammary fat pads, respectively. a, b The percentage of ALDH⁺ BCSCs was determined by ALDEFLUOR assay in tumor cells (CD45⁻CD140b⁻CD31⁻) from tumors of Balb/c mice (a) or C57BL/6N mice (b). Bar graph was presented as the mean of three biologically independent experiments (mean ± SEM). c, d PMN-MDSCs, sorted from pSIN-/CCL20-overexpressing 4T1 or Py8119 cell allograft tumors, were labeled as pSIN^PMN or CCL20^PMN in Figures and as pSIN-PMN-MDSCs or CCL20-modulated PMN-MDSCs in main text. 4T1 or Py8119 cells were co-cultured with pSIN-/CCL20-modulated PMN-MDSCs for 3 days. The percentage of ALDH⁺ BCSCs was determined by ALDEFLUOR assay in 4T1 (c) or Py8119 cells (d). Bar graph was presented as the mean of three biologically independent experiments (mean ± SEM). e, f 4T1 or Py8119 cells were co-cultured with pSIN-/CCL20-modulated PMN-MDSCs for 3 days. The mRNA expression levels of several stemness-related genes were analyzed in 4T1 (e) or Py8119 cells (f) by qRT-PCR. Bar graph was presented as the mean of three biologically independent experiments (mean ± SEM). g–i Self-renewal capability was determined by forming both primary mammospheres (g, n = 7 for each group) and secondary mammospheres (h, n = 4 for each group) in 4T1 cells mix-cultured with pSIN-/CCL20-modulated PMN-MDSCs. The average cell number of each secondary mammosphere was shown (i), and bar graph was presented as mean ± SEM. Scale bar, 1 mm. j, k 4T1 cells were mix-cultured with pSIN-/CCL20-modulated PMN-MDSCs for 3 days, and then sorted by flow cytometry and engrafted to the fourth mammary fat pads of Balb/c mice at a limited dilution (n = 3 for each group, two injection sites each mouse, 100 or 500 cells/site). Tumor image was shown (j). The stem cell frequency in tumor tissue was calculated by the limited dilution assay (k). The stem cell frequency and p-value calculation were based on the positive tumor sites. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001