Extended Data Fig. 5: Mouse astrocytes infiltrate p53-null BMs and promote the survival, proliferation and migration of p53-null mouse BC cells in the brain.
a, Fluorescence-based quantification of microglia in the EMT6-derived tumors. Iba1 staining intensity was similar in tumors growing from Trp53-null and Trp53-WT BC cells. Two-sided t-test p = 0.08. Tumor sections evaluated: Trp53-WT n = 9, Trp53-null n = 9. Mice n = 3. b, Fluorescence-based quantification of endothelial cells in the EMT6-derived tumors. CD31 staining intensity was similar in tumors growing from Trp53-null and Trp53-WT BC cells. Two-sided t-test p = 0.23, Tumor sections evaluated: Trp53-WT n = 11, Trp53-null n = 13. Mice n = 3. c, Live imaging-based proliferation curves of EMT6 BC cells co-cultured with mouse astrocytes. The effect of co-culture on the growth of Trp53-null BC cells is stronger than that of Trp53-WT cells. Two-sided t-test, Trp53-null vs. Trp53-WT: 48 h **p = 0.002, 60 h ***p = 7 × 10−4; 72 h ****p = 2 × 10−5. Trp53-WT n = 8, Trp53-null n = 8. Error bars, mean ± s.e.m. d, Live imaging-based proliferation curves of BC EMT6 cells cultured with ACM. The effect of ACM on the growth of Trp53-null BC cells is stronger than its effect on Trp53-WT cells. Two-sided t-test, Trp53-null vs. Trp53-WT: 24 h ****p = 1.2 × 10−10; 36 h ****p = 1 × 10−10; ****p, 48 h ****p = 4.5 × 10−7; 60 h ****p = 1 × 10−5; 72 h ***p = 2 × 10−4. Trp53-WT n = 7, Trp53-null n = 7. Error bars, mean ± s.e.m. e, Live imaging-based proliferation curves of EMT6 BC cells cultured with ACM. The effect of ACM on the growth of Trp53-null BC cells is stronger than its effect on Trp53-WT cells (shown as cell numbers). Two-sided t-test, Trp53-null vs. Trp53-WT, 60 h *p = 0.04, 72 h *p = 0.02. Error bars, mean ± s.e.m. f, Flow cytometry analysis of BC cells cultured with ACM. The fraction of cells in apoptosis is lower in Trp53-null than in Trp53-WT cells. Fraction of AnnexinV+ cells is shown as fold change relative to SFM controls. Two-sided t-test, Trp53-null vs. Trp53-WT, **p = 0.01, n = 4. See Supplementary Fig. 16a. g, Transwell migration assay to assess the migration of BC cells towards ACM. BC cells were seeded in SFM in the upper compartment and ACM or astrocytes were placed in the lower compartment. h, Quantification of cell migration in the transwell assay after 20 h of co-culture of BC cells and astrocytes, showing all conditions analyzed (see Fig. 3l for relative ACM/SF values). The effect of astrocytes on cell migration was stronger in Trp53-null cells. Two-sided t-test, Trp53-WT SFM vs. Trp53-WT astrocytes ***p = 9 × 10−4; Trp53-null SFM vs. Trp53-null astrocytes ****p = 2.5 × 10−7; Trp53-WT astrocytes vs. Trp53-null astrocytes ****p = 1.7 × 10−6. i, Quantification of cell migration in the transwell assay after 20 h of co-culture of BC cells and ACM. The effect of ACM on cell migration was significantly stronger in Trp53-null cells. Two-sided t-test, Trp53-null SFM vs. Trp53-null ACM p = 0.004, Trp53-WT ACM vs. Trp53-null ACM ****p = 6 × 10−8. The fold increase of migrating cells in ACM over SFM was significant at **p = 0.005. Fields counted TP53-WT n = 17, TP53-null n = 12. j, Live imaging-based quantification of the effect of ACM on cell velocity of Trp53-WT and Trp53-null cells. Cell velocity shown as fold increase in ACM over SFM. Two-sided t-test ****p = 1 × 10−6. Each data point represents a velocity measurement; n = 47. Boxplots: bar = median; box = 25–75th percentile; whiskers = range.
