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. 2021 Dec 15;12:7300. doi: 10.1038/s41467-021-27308-2

Fig. 3. Macrophages regulate stemness in breast cancer cells.

Fig. 3

a Intravital microscopy image of a CSC (arrow) in contact with a macrophage in primary tumor in a living mouse. b Left panel: representative images of fixed-frozen primary tumor tissue showing cancer cell (tdTomato, red), CSC (SORE6+, green), macrophage (Iba1, white), and nuclei (DAPI, blue). Right panel: same as left but lacking tdTomato and DAPI channels, showing CSCs in contact with macrophages (oval), or not in contact with macrophage (square). c Quantification of % CSCs and % non-CSCs in direct contact with macrophages or not in live tissue. n = 42 CSCs from 17 fields of 340 × 340 µm2 in 4 mice, n = 602 non-CSCs from 11 fields of 340 × 340 µm2 in 3 mice; two-sided Fisher’s exact test, ****p < 0.0001. d Quantification of changes in macrophage density in live mammary tumor over time after control PBS liposome or clodronate liposome treatment. Vertical arrows indicate the treatment days. n = 7 fields of 512 × 512 µm2 each for control (3 mice) and clodronate (3 mice) treatments. Two-sided Tukey’s multiple comparisons test; p-values for control: day0 vs. day1 = 0.44, day0 vs. day2 = 0.67; and for clodronate: day0 vs. day1 = 0.09, day0 vs. day2(clodronate) = 0.0005. e Quantification of changes in SORE6+ cells in live mammary tumor over time after control PBS liposome or clodronate liposome treatment. n = 7 fields of 512 × 512 µm2 each for control (3 mice) and clodronate (3 mice) treatments. Two-sided Tukey’s multiple comparisons test; p-values for control: day0 vs. day1 = 0.76, day0 vs. day2 = 0.16; and for clodronate: day0 vs. day1 = 0.007, day0 vs. day2 = 0.0007. f Images of PyMT primary tumor tissues treated with either PBS liposomes (control) or clodronate liposomes and stained with Sox9 antibody (green) and DAPI (blue). Image shows Sox9Hi cells. g Quantification of changes in macrophage density in fixed PyMT mammary tumor after PBS (control) or clodronate liposomes treatment every 2 days for 2 weeks. n = 11 (control) and 6 (clodronate) fields of 2–3 mm2 tumor tissue from 5 mice (control) and 4 mice (clodronate); two-sided Mann–Whitney test, ***p = 0.0002. h Quantification of changes in Sox9Hi cells in fixed PyMT mammary tumor after PBS (control) or clodronate liposomes treatment every 2 days for 2 weeks. n = 17 (control) and 11 (clodronate) fields of 1000 × 500 µm2 tumor tissue from 5 mice (control) and 4 mice (clodronate); Mann–Whitney two-sided test, *p = 0.0221. c–e, g, h The boxes indicate 25th–75th percentile interquartile range (IQR) and central line indicates the median. Top and bottom whiskers were plotted using Tukey’s method and extend to 75th percentile + 1.5 × IQR and 25th percentile − 1.5 × IQR, respectively. Points below and above the whiskers are drawn as individual dots.