1. Volume 21, Issue 8, Pages 2212-2222 (November 2017)
CCR5 Directs the Mobilization of CD11b+Gr1+Ly6Clow Polymorphonuclear Myeloid Cells from the Bone Marrow to the Blood to Support Tumor Development 
Elias Hawila, Hila Razon, Gizi Wildbaum, Carolin Blattner, Yair Sapir, Yuval Shaked, Viktor Umansky, Nathan Karin 
Cell Reports 
Volume 21, Issue 8, Pages (November 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 2212-2222DOI: (10.1016/j.celrep.2017.10.104)
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3. Figure 1
CCR5 Ligands Suppress Tumor Growth by a Direct Effect on CCR5+ Tumor Cells and also via CCR5+ Non-malignant Endogenous Cells
CCR5+/+ C57BL/6 mice (WT) and CCR5−/− C57BL/6 mice (10 per group) were administered with 7 × 106 TRAMP C1-luc cells. Twenty-five days later, five mice from each group were repeatedly administered (twice a week) with 200 μg mCCR5-Ig, isotype-matched control murine IgG (mIgG) or PBS and monitored for the progression of the primary tumor. After 60 days, the mice were sacrificed.
(A) Imaging of primary tumor 60 days post-entrapment as recorded by the charge-coupled device camera (IVIS). Representative mice (left) and analyses of all mice (right) are shown (three experiments, five mice per group; each dot represents a single mouse).
(B) Histological analyses of the primary tumor (day 60) with PCNA staining using anti-mouse primary antibodies and MaxHetero rat on mouse HRP detection kit or Max Hetero rabbit on mouse detection AP kit using standard protocols. Representative slides (left) and cumulative immunohistological data (right) are shown.
(C) H&E staining marked N for necrotic area or V for viable tissue. See also Figure S2
(D) Frozen sections were prepared from tumors and stained for CD31 (an endothelial cell marker) indicating the angiogenesis level of the tumors. Representative slides (upper) and cumulative immunohistological data (lower) are shown.
(B and D) Each dot in the graphs is an average of eight sections per mouse. Each panel represents three biological repeats of five mice (total n = 15).
Values were determined using a one-way ANOVA test using Tukey’s multiple comparisons test (∗∗∗p < 0.001, ∗∗∗∗p < ).
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4. Figure 2
CCR5 Is Essential for the Accumulation of CD11b+Gr1+ Myeloid Cells at the Tumor Site
Ten CCR5+/+ C57BL/6 mice (WT) and ten CCR5−/− C57BL/6 mice were administered with 7 × 106 TRAMP C1-luc cells. Twenty-five days later, five mice of each group were administered, twice a week, with 200 μg murine (m)CCR5-Ig, isotype-matched control mIgG or PBS and monitored for the development of the primary tumor as described in the legend to Figure 1. After 60 days, the mice were sacrificed.
(A) Immunohistological analysis of F4/80+ cells.
(B) Immunohistological analysis of Gr1+ cells. Representative staining (upper) and cumulative immunohistological data (lower) are shown. Each dot in the graph is an average of eight sections per mouse. Each panel represents three biological repeats of five mice (total n = 15). Values were determined using a one-way ANOVA test using Tukey’s multiple comparisons test (∗∗∗p < 0.001, ∗∗∗∗p < ).
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5. Figure 3
Adoptive Transfer of BM Cells from WT to CCR5−/− Mice Restores the Accumulation of Gr1+ Cells at the Tumor Site and Enhances Tumor Development
WT or CCR5−/− recipient mice (10 per group) were lethally irradiated and transplanted with 1 × 107 BM cells from WT or CCR5−/− mice (day 0). On day 30, recipient mice were administered with 7 × 106 TRAMP C1-luc cells.
(A) Imaging of the primary tumor at day 90 recorded by the charge-coupled device camera (IVIS). Representative mice (left) and cumulative imaging data (right) are shown.
(B) Frozen tumor sections were stained with antibodies for CD31 (endothelial cell marker).
(C) Immunohistological analysis of Gr1+ cells. Representative staining (upper) and the cumulative immunohistological data (lower) are shown. Each dot in the graph is an average of eight sections per mouse. Each panel represents three biological repeats of five mice (total n = 15).
(D) Staining with H&E and marked N for necrotic area or V for viable tissue. See also Figure S2.
Values were determined using a one-way ANOVA test using Tukey’s multiple comparisons test (∗∗∗∗p < 0.001, ∗∗∗∗∗p < ).
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6. Figure 4
CCR5+ Directs the Migratory Properties and Induces Suppressive Functions in MDSCs
CCR5+/+ C57BL/6 mice (WT) were administered with 7 × 106 TRAMP C1-luc cells. Twenty-five days later, one-half of the mice were repeatedly administered (twice a week) with 200 μg mCCR5-Ig and monitored for the progression of the primary tumor.
(A) The primary tumor was measured using a caliper.
(B) Tumor weight.
(C and D) After 60 days, the mice were sacrificed, and bone marrow, blood, spleen, and tumor cells were analyzed by FACS for the percentage of CD11b+Gr1+ cells (C) and CCR5+ MDSCs in the tumor (D).
(E) ARG1 expression on CCR5+ or CCR5− MDSCs in the tumor, blood, and bone marrow of tumor-bearing mice.
(F) CCR5+ MDSC cells were separated from tumors and incubated with naive CD4+ T cells in different ratios (1:1, 1:2, 1:4, and 1:8) and compared to non-activated CD4 T cells or activated CD4 without MDSCs. CD4+ T cell proliferation was measured using CFSE labeling after 72 hr.
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7. Figure 5
Effects of CCR5 Ligands on the Migration of CCR5+CD11b+Gr1+ Cells In Vitro
BM cells were harvested from five WT and five CCR5−/− mice.
(A) Representative dot plots showing CCR5+CD11b+Gr1+.
(B) Gr1+ cells from the BM of WT and CCR5−/− mice were separated using anti-PE magnetic beads. A migration assay was performed using 3-μm Transwell inserts. The migration index was calculated as a ratio of the number of migrated cells treated with chemokines to that in non-treated wells (means ± SDs). Results are shown as data of one representative experiment of three. p values were determined using a one-way ANOVA test using Tukey’s multiple comparisons test or a two-way ANOVA test using Bonferroni’s multiple comparisons test (∗∗∗∗p < ).
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8. Figure 6
CCR5 Ligands Induce Migration of CD11b+Gr1+ Myeloid Cells from the BM to the Peripheral Blood in a CCR5-Dependent Manner
(A and B) WT (A) and CCR5−/− (B) mice were injected with 2 μg CCL2, CCL3, CCL4, CCL5, or PBS (control). Peripheral blood was collected from the tail vein at different treatment time points (1, 2, 4, 6, and 10 hr). Cells were stained with antibodies for Gr1 and CD11b and analyzed by flow cytometry. The left graphs represent results of a representative experiment (n = 4 mice). The right graphs show cumulative data of four different experiments. Each dot in the scatterplot represents a single group (n = 4) in each experiment. The plot shows and analyzes a single time point (2 hr). Values were determined using a one-way ANOVA test using Tukey’s multiple comparisons test (∗∗∗∗p < ). See also Figure S3.
(C–E) WT mice were injected (i.v.) with the combination of CCL3, CCL4, and CCL5 (2 μg each) or PBS (control). Bone marrow (C), peripheral blood (D), and spleen (E) cells were collected 1, 2, 6, 10, or 24 hr after the injection. Cells were stained with antibodies for Gr1 and CD11b and analyzed by flow cytometry. The graphs show cumulative data from five independent experiments. (C and E) p values were determined using two-way ANOVA test using Tukey’s multiple comparisons test. (D) p values were determined using two-way ANOVA test using Bonferroni’s multiple comparisons test.
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < See also Figure S4.
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9. Figure 7
CCR5 Directs the Mobilization of CD11b+Gr1+Ly6Clow Myeloid Cells from the BM to the Peripheral Blood
WT mice were injected (i.v.) with the combination of CCL3, CCL4, and CCL5 (2 μg each) or CCL2 (2 μg) or PBS. BM and peripheral blood cells were collected before (naive mice) and, 2 hr upon the treatment, stained with antibodies for Ly6C, Gr1, and CD11b and analyzed by flow cytometry.
(A and B) Results show the frequency of CD11b+Gr1+Ly6Clow (A) or CD11b+Gr1+Ly6Chigh (B) cells among total leukocytes in the peripheral blood (n = 5 mice). Values were determined using a one-way ANOVA test using Tukey’s multiple comparisons test (∗∗∗∗p < ). See also Figure S5.
(C and D) Graphs represents the frequency of CD11b+Gr1+Ly6Clow (C) or CD11b+Gr1+Ly6Chigh (D) cells among total leukocytes in the BM (n = 5 mice). Values were determined using a one-way analysis of variance (ANOVA) test using Tukey’s multiple comparisons test. For (C), ∗p = 0.02, ∗∗p < 0.01, and ∗∗∗p < For (D), ∗p = 0.04 and ∗∗p < 0.01.
(E–G) CCR5+/+ C57BL/6 mice (WT) were administered with 7 × 106 TRAMP C1-luc cells. Twenty-five days later, one-half of the mice were repeatedly administered (twice a week) with 200 μg mCCR5-Ig. After 60 days, the mice were sacrificed, and bone marrow, blood, spleen, and tumor cells were analyzed (FACS) for the percentage of PMN-MDSC (E), Mo-MDSC (F), or CCR5+ expression (G).
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