PMC

Loss of FBXW7 in BMSCs results in accumulation of NICD1 and increased secretion of CCL2, which in turn promotes recruitment of Mo-MDSCs and macrophages. These cells then promote the growth of tumors that have already colonized the lungs. TAM, tumor-associated macrophage.

Representative immunohistofluorescence staining (white) for TCRβ (A), B220 (B), Ly6G (C), Ly6C (D), F4/80 (E), and FSP (F) for lung sections from WT mice reconstituted with CAG-EGFP Fbxw7^fl/fl (n = 10 [A, B, D, and E]; 9 [C]; 11 [F]) or CAG-EGFP Mx1-Cre Fbxw7^Δ/Δ (n = 10 [A, C, and E]; 12 [B]; 14 [D]; 11 [F]) BM cells and subjected to orthotopic transplantation with tdTomato-labeled E0771 cells (20 days before analysis) as in Figure 2H. Intrinsic fluorescence of EGFP (green), tdTomato (red), and Hoechst 33258 (blue) was also imaged. Higher-magnification images (×2) are shown in the insets. Scale bars: 100 μm. The percentage of EGFP⁺ BMDCs positive for each marker in tumor-surrounding and nonsurrounding regions was quantified; horizontal bars indicate means. *P < 0.05, ***P < 0.001, 1-way ANOVA and Bonferroni test.

(A and B) Kaplan-Meier curves for overall (A) and disease-free (B) survival of breast cancer patients (n = 406) classified according to the abundance of FBXW7 mRNA in peripheral blood. (C–E) Kaplan-Meier curves for disease-free survival of breast cancer patients with luminal (C), HER2⁺ (D), or triple-negative ER^–PR^–HER2^– (E) tumors classified according to the abundance of FBXW7 mRNA in peripheral blood. *P < 0.05, **P < 0.01, log-rank test. (F) Representative immunohistochemical staining patterns for FBXW7 in breast cancer patients: positive in both primary tumor cells and stroma, positive in tumor cells only, positive in stroma only, or negative in both tumor cells and stroma. T, tumor cells; S, surrounding stromal cells. Scale bars: 100 μm. (G and H) Mosaic plots summarizing the abundance of FBXW7 mRNA in peripheral blood and FBXW7 expression in tumor cells (G) and surrounding stroma (H) for the indicated numbers of breast cancer patients (n = 22). P values for the association between these parameters were calculated by χ² test. (I) Correlation between the abundance of FBXW7 mRNA in peripheral blood and the serum CCL2 concentration in breast cancer patients (n = 57).

(A and B) B16F10 cells were transplanted into Fbxw7^+/fl (n = 7), Fbxw7^fl/fl (n = 8), Mx1-Cre Fbxw7^+/Δ (n = 11), and Mx1-Cre Fbxw7^Δ/Δ (n = 12) mice. The gross appearance of the lungs (A) and their occupancy by tumor colonies (B) were examined. Horizontal bars in B indicate mean values. (C) Kaplan-Meier survival curves for Fbxw7^fl/fl (n = 9) and Mx1-Cre Fbxw7^Δ/Δ (n = 10) mice after injection of B16F10 cells. (D and E) LLC cells were transplanted into Fbxw7^+/fl (n = 4), Fbxw7^fl/fl (n = 5), Mx1-Cre Fbxw7^+/Δ (n = 5), and Mx1-Cre Fbxw7^Δ/Δ (n = 12) mice. (F and G) B16F1 cells were transplanted into Fbxw7^+/fl (n = 9), Fbxw7^fl/fl (n = 8), Mx1-Cre Fbxw7^+/Δ (n = 8), and Mx1-Cre Fbxw7^Δ/Δ (n = 8) mice. Lungs were subjected to H&E staining (D and F), and their gross weight was determined (E and G). (H–J) Metastasis assays performed in WT mice reconstituted with CAG-EGFP Fbxw7^fl/fl (n = 8) or CAG-EGFP Mx1-Cre Fbxw7^Δ/Δ (n = 7) donor BM. (K–M) Metastasis assays performed in Fbxw7^fl/fl (n = 10) or Mx1-Cre Fbxw7^Δ/Δ (n = 12) mice reconstituted with WT donor BM. Schematic representation (H and K), gross appearance of the lungs (I and L), and their occupancy by tumor colonies (J and M) are shown. Scale bars: 10 mm (A, I, and L); 2 mm (D and F). Horizontal bars in B, E, G, J, and M indicate means. ***P < 0.001, 1-way ANOVA and Bonferroni test (B, E, and G) or 2-tailed Student’s t test (J).

(A) Immunoblot analysis of FBXW7 substrates in the indicated BMSCs. (B) Relative abundance of Ccl2 mRNA in WT BMSCs infected with retroviruses encoding NICD1, c-MYC, or KLF5. (C) Luciferase assay for the Ccl2 gene in BMSCs infected with retroviruses for NICD1, c-MYC, or KLF5. (D) Relative abundance of Ccl2 mRNA in CAG-Cre-ER^T2 Fbxw7^Δ/Δ BMSCs incubated with DAPT. (E) WT and mutant forms of the mouse Ccl2 gene promoter fused to the firefly luciferase gene. Consensus binding sequences for NOTCH–RBP-Jκ are shown in bold. Proximal and distal amplicons in G are indicated. (F) Luciferase assay for the Ccl2 gene in CAG-Cre-ER^T2 Fbxw7^Δ/Δ BMSCs. (G) ChIP analysis of the Ccl2 gene promoter. Immunoprecipitation was performed with antibodies against NOTCH1 or with control IgG. (H and I) Intravenous transplantation with B16F10 cells for Fbxw7^fl/fl (n = 8), Fbxw7^fl/fl RbpJκ^fl/fl (n = 11), Mx1-Cre Fbxw7^Δ/Δ (n = 5), and Mx1-Cre Fbxw7^Δ/Δ RbpJκ^Δ/Δ (n = 8) mice. (J and K) Intravenous transplantation with B16F10 cells for Fbxw7^fl/fl (n = 8), Fbxw7^fl/fl c-Myc^fl/fl (n = 7), Mx1-Cre Fbxw7^Δ/Δ (n = 6), and Mx1-Cre Fbxw7^Δ/Δ c-Myc^Δ/Δ (n = 8) mice. Gross appearance of the lungs (H and J) and their occupancy by B16F10 colonies (I and K) are shown. (L) Serum concentration of CCL2, determined by ELISA. Scale bars: 10 mm (H and J). Data are mean ± SD (n = 3) (B–D, F, G, and L); horizontal bars in I and K indicate means. **P < 0.01, ***P < 0.001, 1-way ANOVA and Bonferroni test (B–D, I, K, and L).

(A) Representative immunohistofluorescence staining of Ly6C and FSP in lung sections from WT mice reconstituted with indicated BM cells and subjected to orthotopic transplantation with tdTomato-labeled E0771 cells (20 days before analysis). (B) Genomic PCR analysis of BMSCs from the indicated mice incubated in the absence or presence of 10 μM tamoxifen. The positions of amplified fragments corresponding to floxed and exon 5–deleted (ΔE5) Fbxw7 alleles are indicated. (C) Relative abundance of Ccl2 mRNA in BMSCs from the indicated mice. (D) Concentration of CCL2 released into culture supernatants from the indicated BMSCs. (E) Relative abundance of Ccl2 mRNA in CAG-Cre-ER^T2 Fbxw7^Δ/Δ BMSCs infected with retroviruses encoding WT or ΔF mutant forms of FBXW7α or with the empty virus (Mock). (F) Relative abundance of Ccl2 mRNA in BMSCs from Fbxw7^fl/fl or CAG-Cre-ER^T2 Fbxw7^fl/fl mice treated with tamoxifen and subjected to RNAi with shRNA vectors targeting EGFP (control) or CCL2. (G and H) Gross appearance of the lungs (G) and their occupancy by visible B16F10 colonies (H) for WT mice 2 weeks after injection both with B16F10 cells and with BMSCs isolated from the indicated mice and treated as in F (n = 10 per group). Scale bars: 10 μm (A), 10 mm (G). Data are mean ± SD (C–F); horizontal bars in H indicate means. *P < 0.05, ***P < 0.001, 1-way ANOVA and Bonferroni test (C, E, F, and H) or 2-tailed Student’s t test (D).

(A and B) E0771 cells were transplanted into the mammary fat pad of Fbxw7^fl/fl (n = 11) and Mx1-Cre Fbxw7^Δ/Δ (n = 13) mice. Primary tumor gross appearance after 32 days (A) and volume at the indicated times (B) are shown. (C–G) E0771 cells were transplanted into the mammary fat pad of Fbxw7^fl/fl (n = 12) and Mx1-Cre Fbxw7^Δ/Δ (n = 16) mice. After 32 days, lungs were subjected to H&E staining (C), and their gross weight was determined (D). Tumor total area (E), number of tumor nodules (F), and average tumor area (G) were calculated from the stained lung sections. (H–J) WT mice were reconstituted with BM cells of the indicated mice and subjected to orthotopic transplantation with tdTomato-labeled E0771 cells. Lungs were subjected to fluorescence microscopy for detection of BMDCs (green), tumor cells (red), and cell nuclei (Hoechst 33238) (H), and the number of tumor nodules (I) and average tumor area (J) were determined 16 (n = 8 per group) or 20 (n = 9 per group) days after tumor cell transplantation. Scale bars: 2 mm (C); 100 μm (H). Data in B are mean ± SEM; horizontal bars in D–G and I indicate means; box and whisker plots in J depict the smallest value, lower quartile, median, upper quartile, and largest value. **P < 0.01, ***P < 0.001, 2-tailed Student’s t test (B, D–G, I, and J).

(A) Serum cytokine levels in Fbxw7^fl/fl and Mx1-Cre Fbxw7^Δ/Δ mice after orthotopic transplantation of tdTomato-labeled E0771 cells or in nontransplant controls. Optical densities for cytokines are indicated according to the color scale. See also Supplemental Figure 5. (B) Serum concentration of CCL2, determined by ELISA, in the indicated mice transplanted with tdTomato-labeled E0771 cells. Data are mean ± SD (n = 4 per group). (C and D) Gross appearance of the lungs (C) and their occupancy by visible B16F10 tumor colonies (D) for Fbxw7^fl/fl (n = 5 [not treated]; 8 [treated]) and Mx1-Cre Fbxw7^Δ/Δ (n = 8 [not treated]; 6 [treated]) mice injected with B16F10 cells and treated or not with the CCR2 antagonist propagermanium. (E–H) WT mice were reconstituted with CAG-EGFP Mx1-Cre Fbxw7^Δ/Δ BM cells, subjected to orthotopic transplantation with tdTomato-labeled E0771 cells, and treated or not with propagermanium (n = 7). At 20 days after tumor cell transplantation, lungs were subjected to fluorescence microscopy (E), and number of tumor nodules (F) and average tumor area (G) were determined. (H) The percentage of EGFP⁺ BMDCs positive for Ly6C in the tumor-bearing lungs of mice as in E–G was determined by immunohistofluorescence analysis. Scale bars: 10 mm (C); 100 μm (E). Horizontal bars in D, F, and H indicate means; box and whisker plots in G depict the smallest value, lower quartile, median, upper quartile, and largest value. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA and Bonferroni test (B and D) or 2-tailed Student’s t test (F–H).