Fig. 2: Epigenetic regulation of TFF3 maintains a dormancy-associated phenotype.

a Venn plot: A Venn plot was generated by identifying common genes in upregulated FD/FC or TD/TC gene sets with extracellular space gene sets. The genes were considered with fold change ≥2 and false discovery rate (FDR)/Q value ≤ 0.001. Cells were cultured in 10% FBS containing medium (b) Heatmap: A heatmap was constructed to cluster gene expression (mRNA TPM) of the six common genes (identified in a) in control (FC/TC), dormancy-like (FD/TD), or relapse (FR/TR) cells. The data were standardized using the z-score method in the row direction. c Volcano plot: A volcano plot was created to show differentially expressed genes (DEGs) between FD/FC or TD/TC cells with fold change ≥2 and FDR/Q value ≤ 0.001. The X-axis represents Log₂ transformed difference multiplier values, and the Y-axis represents -Log₁₀ transformed significance (Q) values. Red represents up-regulated DEGs, blue represents down-regulated DEGs, and gray represents non-DEGs. d Western blot analysis: The densitometric analysis of protein blots was performed using ImageJ software (https://imagej.nih.gov/ij/) to assess the statistical changes between the groups in Western blot analysis in Fig. S11c (Fig. S34). Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparisons test (n = 3). e IHC: The expression of TFF3 was assessed in resected xenografts of FC, FD, FR, TC, TD, or TR groups using IHC and the immunoreactive score of each representative figure is presented in their bottom left corner. The images were captured at a magnification of ×200, with a scale bar of 20 μm. f ELISA: ELISA was performed for host animal serum levels of human (h)TFF3 (derived from xenografts) (n = 3). The FD or FR groups were subcutaneously treated with 250 mg/kg Fulvestrant per week and the TD or TR groups were treated intraperitoneally with 30 mg/kg of Tamoxifen twice a week for 4 weeks. The FC and TC groups received the corresponding vehicles for 4 weeks. Blood was collected weekly during administration. Serum hTFF3 concentration (pg/ml) normalized to xenograft volume (mm³) is represented in a folding line chart. The right bar chart represents the normalized serum hTFF3 levels of host animal at the endpoint (E). Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparisons test. g ELISA: ELISA was used to determine the serum levels of human (h)TFF3 in mice injected with FC/FD/FR and TC/TD/TR cells through the tail vein (n = 6). All measurements were normalized to the average of hTFF3 levels in the corresponding control groups as relative serum hTFF3. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparisons test. The symbols (*) indicate statistical significance compared to the corresponding control group. p < 0.05(*), p < 0.01(**) and p < 0.001(***) were considered statistically significant. p < 0.0001, unless indicated otherwise.