Figure 7.
Figure 7. Increased β-catenin expression inhibits MDSC expansion, activity, and tumor growth. (A) 105 LLC cells were s.c. injected in β-cat.CA or control mice (CTR), and tumor growth was followed for 14 d. Bone marrow, spleen, and tumors were then analyzed by FACS using anti–Gr-1 and CD11b staining to measure the percentage of MDSCs. Mean ± SD (n = 4) are shown. One representative out of two independent experiments is shown. *, P < 0.05; **, P < 0.01. (B) T cell proliferation assay. MDSCs were isolated from CTR and β-cat.CA mice, co-cultured with CFSE-labeled splenocytes from WT mice (1:5 and 1:1 ratios) and stimulated with anti-CD3 (10 µg/ml) for 3 d. Bar graphs show mean ± SD of three independent experiments. *, P < 0.05. Representative flow cytometric analysis of gated CD8+ T cell proliferation in the presence of CTR- (dashed line) and β.cat.CA-MDSCs (solid line) is also shown. (C) WT mice were lethally irradiated and transplanted with bone marrow cells from PLCγ2cKO, LysM-Cre (CTR), or PLCγ2cKO/β-cat.CA to generate chimeric mice. Western blot analyses show the expression of PLCγ2, β-catenin, p-PKC, p-GSK3β, and β-actin in MDSCs from chimeric mice. 4 wk after BM transplantation, chimeric mice were inoculated s.c. with 105 LLC cells and tumor growth was followed for 14 d. Percentage of MDSCs in bone marrow, spleen, and tumor was analyzed by FACS at time of sacrifice. Mean ± SD (n = 10) are shown. Data are reported from one of two similar independent experiments. **, P < 0.01; ***, P < 0.001. (D and E) 105 LLC cells were s.c. injected in PLCγ2cKO, CTR, or PLCγ2cKO/β-cat.CA mice. 5 d after tumor challenge, the tumor was resected and weighed (D). Percentage of MDSCs from bone marrow, spleen, and tumor were then analyzed by FACS staining (Gr-1+CD11b+, MDSCs) (D). Dissected tumors were stained with anti-CD8 antibody and the percentage of CD8+ T cells was determined by FACS (E). Results represent mean ± SD (n = 3). One representative out of two independent experiments is shown (D and E). *, P < 0.05; **, P < 0.01. (F) T cell proliferation assay. 5 d after LLC tumor inoculation, MDSCs were isolated from CTR, PLCγ2cKO, and PLCγ2cKO/β-cat.CA mice, co-cultured for 3 d with CFSE-labeled splenocytes from WT mice (1:5 and 1:1 ratios), and stimulated with anti-CD3 (10 µg/ml). Bar graphs show mean ± SD of three independent experiments. *, P < 0.05.

Increased β-catenin expression inhibits MDSC expansion, activity, and tumor growth. (A) 105 LLC cells were s.c. injected in β-cat.CA or control mice (CTR), and tumor growth was followed for 14 d. Bone marrow, spleen, and tumors were then analyzed by FACS using anti–Gr-1 and CD11b staining to measure the percentage of MDSCs. Mean ± SD (n = 4) are shown. One representative out of two independent experiments is shown. *, P < 0.05; **, P < 0.01. (B) T cell proliferation assay. MDSCs were isolated from CTR and β-cat.CA mice, co-cultured with CFSE-labeled splenocytes from WT mice (1:5 and 1:1 ratios) and stimulated with anti-CD3 (10 µg/ml) for 3 d. Bar graphs show mean ± SD of three independent experiments. *, P < 0.05. Representative flow cytometric analysis of gated CD8+ T cell proliferation in the presence of CTR- (dashed line) and β.cat.CA-MDSCs (solid line) is also shown. (C) WT mice were lethally irradiated and transplanted with bone marrow cells from PLCγ2cKO, LysM-Cre (CTR), or PLCγ2cKO/β-cat.CA to generate chimeric mice. Western blot analyses show the expression of PLCγ2, β-catenin, p-PKC, p-GSK3β, and β-actin in MDSCs from chimeric mice. 4 wk after BM transplantation, chimeric mice were inoculated s.c. with 105 LLC cells and tumor growth was followed for 14 d. Percentage of MDSCs in bone marrow, spleen, and tumor was analyzed by FACS at time of sacrifice. Mean ± SD (n = 10) are shown. Data are reported from one of two similar independent experiments. **, P < 0.01; ***, P < 0.001. (D and E) 105 LLC cells were s.c. injected in PLCγ2cKO, CTR, or PLCγ2cKO/β-cat.CA mice. 5 d after tumor challenge, the tumor was resected and weighed (D). Percentage of MDSCs from bone marrow, spleen, and tumor were then analyzed by FACS staining (Gr-1+CD11b+, MDSCs) (D). Dissected tumors were stained with anti-CD8 antibody and the percentage of CD8+ T cells was determined by FACS (E). Results represent mean ± SD (n = 3). One representative out of two independent experiments is shown (D and E). *, P < 0.05; **, P < 0.01. (F) T cell proliferation assay. 5 d after LLC tumor inoculation, MDSCs were isolated from CTR, PLCγ2cKO, and PLCγ2cKO/β-cat.CA mice, co-cultured for 3 d with CFSE-labeled splenocytes from WT mice (1:5 and 1:1 ratios), and stimulated with anti-CD3 (10 µg/ml). Bar graphs show mean ± SD of three independent experiments. *, P < 0.05.

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