Fig. 6: CCR1 inhibition reduces Ly6C^- IM recruitment leading to AM expansion and NK cell accumulation in the lung.

A Flow cytometry analysis was performed to quantify the frequency of the MR^-Ly6C⁺/Ly6C^- and MR⁺Ly6C⁺/Ly6C^- subsets of IM in 10⁵ total events in the lungs of the control or CCRi-treated mice. The bars represent the mean cell numbers + SD, n = 5 both at d3 and d21. The differences between the means were assessed by one-way ANOVA followed by Dunnett’s multiple comparison test between total IM of experimental groups and the respective controls, *p < 0.05. Significant differences between the Ly6C⁺ subsets are shown by orange lines with p-values, whereas significant differences between the Ly6C^- subsets of experimental groups and the respective control are indicated by p-values displayed across the blue bars. B The concentrations of the pro-inflammatory CCL2, CCL5 and IL-12 along with the pro-tumour VEGFA were determined by ELISA in the CM of AMJ2-C11 macrophages separated from their d3 or d21 co-culture with B16F10 cells and expressed as percentages of the monoculture concentrations. The bars represent the mean relative concentrations + SD, n = 3. The differences between the means were assessed by independent t-tests. Representative dot plots are showing the frequency of the Ly6C-expressing macrophages from the d3 and d21 co-cultures. C Flow cytometry analysis was performed to quantify the AM compartments following inhibition of CCRs. The frequency of AM in 10⁵ total events was expressed as mean AM numbers + SD, n = 5 per group at d3 and n = 7–11 per group at d21. One-way ANOVA was carried out followed by Dunnett’s multiple comparison test. D The frequency of T-cells, neutrophils and NK cells were quantified by flow cytometry in 10⁵ total events in lung cell suspensions at d21 using antibodies against CD3ε, Gr1 (Ly6G) and NK1.1, respectively. The bars indicate the mean cell numbers + SD, n = 5 per group. One-way ANOVA followed by Dunnett’s multiple comparison tests were performed. E The quantity of F4/80⁺CD11c⁺ AM and NK1.1⁺ NK cells expressed as % of viable cells in late-stage metastatic lungs correlate as shown by the combined d21 data, n = 16. Non-parametric Spearman’s correlation was carried out, p = 0.0002; linear regression with 95% CI. F Luminex assay was performed to determine the concentrations of chemokines in the conditioned medium of ex vivo AM cultures from unchallenged (AM ctrl) and B16F10-challenged (24 h) lungs (AM + tu), n = 3 each. The concentrations were normalised to cell numbers. The bars represent the average concentrations + SD.