Extended Data Fig. 9: FIP4 inhibits malignancy and enhances immunogenicity in tumor cells.

a-b, Transwell migration assay (a) and transwell invasion assay (b) using MDA-MB-231 cells non-treated (control) or treated with FIP4 (50 μM) for 48 h. Representative figures from three independent experiments are shown. The quantification of migrated cells per field is shown in the bar graphs; Scale bar, 50 μm. c, Growth curves of MDA-MB-231 (left) and 4T1 (right) cells non-treated (PBS) or treated with FIP4 (50 μM); n = 3 independent experiments. d, Heatmap and volcano plot illustrating differentially regulated gene expression from RNA-seq analysis in MDA-MB-231 cells treated for 48 h with control PBS or FIP4 (50 μM) (left). Genes upregulated and downregulated are shown in red and blue, respectively (fold change > 2) (right). Values are presented as the log2 of tag counts. e, Gene ontology (GO) functional clustering of significantly changed genes in MDA-MB-231 cells stimulated with FIP4 (50 μM) for 48 h. f, Gene signatures indicating the breast cancer-related genome amplification, mutation of BRCA1 and squamous breast tumor are significantly enriched in control MDA-MB-231 cells and the decreased breast cancer relapse in lung are significantly enriched in MDA-MB-231 cells treated with FIP4 (50 μM) for 48 h; shown by pre-ranked gene set enrichment analysis (GSEA). g, Gene signatures representing increased tumor invasiveness, metastasis and oncogenic transcription were significantly enriched in control MDA-MB-231 cells versus the cells treated with FIP4 (50 μM) for 48 h; shown by pre-ranked gene set enrichment analysis (GSEA). h, The relative tumor volume (left) and tumor weight (right) at the endpoint (day 35) in Fig. 4j. n = 6 mice per group. i, Immunofluorescence microscopy of FOXM1 and DAPI staining of the tumor isolated from mice (left) as in Fig. 4j. Quantified average number of nuclear FOXM1 puncta, the percentages of cells with nuclear puncta and the average puncta size were shown (right); cells were fixed; n = 24 tumor cells pooled from 6 control mice and 6 FIP4-treated mice as in Fig. 4j. Scale bar, 5 μm. j-n, Experimental analysis in vivo: 4T07-met cells (5 × 10⁵ cells per mouse) were tail vein-injected into nude mice (n = 5 mice per group). Mice were also given injections of PBS or FIP4 (10 mg kg ⁻¹; i.p.) per mouse three times one week (j). Lung metastasis was measured by bioluminescence imaging (BLI). Photon flux (k) and representative images (l) in each experimental group followed in time. Representative H&E stained lung sections from each group at week 4 (m). Scale bar, 1 mm. Percentage of lung metastasis area (left), average lung lesion surface area (middle) and number of lung metastasis nodules (right) per H&E stained lung section of all mice from each group at week 4 (n). o, Representative lung nodules from MMTV-PyMT mice injected with control and FIP4 at the endpoint (week 12) in Fig. 4n. Black arrowheads indicate lung metastasis nodules. Scale bar, 2 mm. Data are representative of at least three independent experiments (c, f, i, k, n). Mean ± s.d., statistical analysis was performed using two-way ANOVA (c, k), Wald test (d), Hypergeometric test (e),Weighted Kolmogorov-Smirnov statistic test (f, g) or two-tailed Student’s t-test (h, i, n).

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