Fig 1: Glioma-derived CCL2 or CCL7 is necessary and sufficient for bone marrow-derived CCR2/CX3CR1 cell migration. (A) Graph depicting CCR2+/CX3CR1+ cells migrating to conditioned media of KR158B (n=6) and KR158B CCL2 knockdown (n=6) cells. A condition in which conditioned media was also plated in the top chamber of the transwell plate (100/100) is included to validate that migration is due to chemotaxis rather than chemokinesis. (B) Migration of CCR2+/CX3CR1+ cells to conditioned media in the presence or absence of chemokine-neutralizing antibodies. Migration is disrupted with the addition of high concentrations of single neutralizing antibody. Migration is completely inhibited with a combination of neutralizing antibodies at either low or high concentrations (n=4). No migration was observed to conditioned media of bone marrow-derived RFP-expressing cells from Ccr2RFP/RFP animals (n=3). (C) Graph depicting that migration to exogenous recombinant CCL2 or CCL7 is inhibited through the addition of high concentrations of respective neutralizing antibody (n=4). Two-way ANOVA statistical analysis was conducted (Dunnett’s multiple comparisons test). Differences are compared to the control condition or between cell lines. p-values: 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****).
Fig 2: High CCL2 and CCL7 expression is associated with negative prognosis for patients with glioblastoma. (A–C) Kaplan-Meyer survival curves of GBM patients based on Affymetrix gene expression profiles of (A) CCL2 (B) CCL7 (C) intersection of CCL2 and CCL7 from TCGA database. (D–F) Kaplan-Meyer survival curves of GBM patients based on Illumina Highseq expression profiles of (D) CCL2 (E) CCL7 (F) intersection of CCL2 and CCL7 mined from TCGA database. High and low cohorts are stratified as top and bottom quartiles, respectively. Number at risk indicates surviving patients in each cohort at the respective timepoints of analysis. Log-rank (Mantel-Cox) test was conducted on high vs low expressing cohorts.
Fig 3: Infiltration of CCR2+/CX3CR1+ cells into the glioma is reduced upon combination targeting of CCL2 and CCL7. (A) Treatment schematic for targeting CCL2 and CCL7 in KR158B or KR158B CCL7 KD gliomas. Anti-CCL2 antibody loading dose of 100μg was administered 3 days post implantation (DPI). Subsequent maintenance doses of 50μg were administered twice weekly. Non-immune IgG was used as a control. A total of 4 arms were evaluated: KR158B treated with IgG control, KR158B CCL7 KD treated with IgG control, KR158B treated with anti-CCL2 antibody, and KR158B CCL7 KD treated with anti-CCL2 antibody. (B) Graph depicting percentage of infiltrating live, CD45+, CD11b+, CCR2+ only cells within the tumor. (C) live, CD45+, CD11b+, CCR2+/CX3CR1+ cells within the tumor (D) live, CD45+, CD11b+, CX3CR1+ only cells within the tumor. (E) Representative flow plot for panels B-D depicting reduction of CCR2+/CX3CR1+ population within the tumor (n=5-6 per arm). Example gating strategy can be found in Supplementary Figure 11A . Two-way ANOVA statistical analysis was conducted (Dunnett’s multiple comparisons test). Differences are compared to the control condition or between cell lines. p-values: 0.0332(*), 0.0021(**), 0.0002(***).
Fig 4: Bone marrow-derived CCR2+/CX3CR1+ cells migrate to recombinant CCL2 and CCL7 through CCR2. (A) Experimental design of transwell migration assays. Graphic (Created with BioRender.com) depicting assay preparation in which whole bone marrow is plated in the top chamber of the transwell migration plate (left). The bottom chamber contains either recombinant chemokine protein or conditioned media. Representative flow plot depicting the population of CCR2+/CX3CR1+ cells quantified (right). (B) Migration to recombinant CCL2 (n=7) and CCL7 (n=4) of CCR2+/CX3CR1+ cells derived from tumor-bearing animals. Graph also depicts no migration to recombinant CCL2 of bone marrow-derived RFP-expressing cells from Ccr2RFP/RFP animals. (C) Migration to recombinant CCL2 and CCL7 of CCR2/CX3CR1-expressing cells derived from naïve animals (n=4). A condition in which chemokine was also plated in the top chamber of the transwell plate (30/30) is included to validate that migration is due to chemotaxis rather than chemokinesis. Two-way ANOVA statistical analysis was conducted (Dunnett’s multiple comparisons test). Differences are compared to the control (0) condition. p-values: 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****).
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