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Blood, Vol. 111, Issue 12, 5457-5466, June 15, 2008
Chemokine-mediated rapid turnover of myeloid-derived suppressor cells in tumor-bearing mice
Blood Sawanobori et al.
111: 5457
Supplemental materials for: Sawanobori et al
Files in this Data Supplement:
- Table S1. Primers and probes used in real-time RT-PCR (PDF, 674 KB)
- Figure S1. Intratumoral distribution of macrophages and neutrophils (JPG, 207 KB)
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Serial sections of 3LL tumors with a diameter of 6-8 mm were subjected to H&E staining (A) or immunofluorescent staining with antibodies to CD11b (green), F4/80 (red), Type IV collagen (blue) (B) or CD11b (green), MMP9 (red), Type IV collagen (blue) (C). Representative image of 3 mice. Original magnification, (×200)
- Figure S2. Induction of immature myeloid cells in the spleen of tumor-bearing mice in the terminal phase (JPG, 140 KB)
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(A) Expression profile of Gr-1 and Ly-6C in CD11b+ cells from BM, spleen and tumor of untreated or tumor-bearing mice on day 20 or 40 after tumor inoculation. (B) Cytospin samples of CD11b+Gr-1hiLy-6Cint/dull (R1), CD11b+Gr-1int/dullLy-6Cint (R2) and CD11b+Gr-1int/dullLy-6Chi (R3) cells from spleen of tumor-bearing mice on day 40 were subjected to Giemsa staining or esterase double staining (R2 small window). Original magnification, (×1200). Note that CD11b+Gr-1int/dullLy-6Cint/dull cells contain immature myeloid cells at various differentiation stages. Arrow head indicates promyelocyte, arrow indicates myelocyte.
- Figure S3. Chemokine receptor expression on neutrophils and macrophages (JPG, 83.8 KB)
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(A) Chemokine receptor expression on CD11b+Gr-1hiLy-6Cint neutrophils (upper) and CD11b+Gr-1int/dullLy-6Chi macrophages (lower) were analyzed using mAb to CCR5, CXCR2 and CXCR4 or CX3CR1+/gfp reporter mice. Shaded histograms indicate background staining with isotype control for CCR5, CXCR2 and CXCR4 or background fluorescence for CX3CR1, solid lines staining with mAbs or reporter GFP expression, and dashed lines in CCR5 indicate CCR5−/− mice as a negative control. (B) mRNA expression of CCR1 and CCR2 by BM monocytes and tumor infiltrating CD11b+ cells, macrophages and neutrophils was analyzed by RT-PCR. Data are obtained from pooled cells from 5 mice. Representative data from 2 independent experiments.
- Figure S4. Regulation of the dynamics of MDSCs subpopulations by CCR2 (JPG, 120 KB)
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CCR2+/+ or CCR2−/− mice were inoculated with 3LL and the percentage of CD11b+Gr-1int/dullLy-6Chi monocytic cells (A) and CD11b+Gr-1hiLy-6Cint neutrophils (B) in CD45+ leukocytes in tumor, spleen, blood and bone marrow were analyzed at various time points. Each time point represents the mean + SD of 3 mice. *p< .05, **p< .01 (C) Equal number of CCR2+/+CD45.1+CD45.2+ and CCR2−/−CD45.1−CD45.2+ bone marrow cells were i.v. injected into CCR2+/+CD45.1+CD45.2− recipient mice, which had been subcutaneously inoculated with tumor cells 7 days before transfer. Twenty-four hours later, monocytic cells from the tumor, spleen, blood, and BM were analyzed by a flow cytometry. (D) Representative flow cytometry profiles of CD11b+Gr-1int/dullLy-6Chi monocytic cells. Black and red gates indicates CCR2+/+ and CCR2−/− donor derived cells, respectively. Numbers in each plot indicate mean percentage of each colored gate. (E) Ratio of CCR2−/−/CCR2+/+ donor cells in each compartment. Graph represents the mean + SD of 3 mice.
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