Fig 2: Activation of p38/MAPK, p65/NF-κB, and STAT3 in monocytes induced by S100A9 or HMGB1. (A) Representative histograms and cumulative data for RAGE and TLR4 expression in monocytes treated with HMGB1 or S100A9 for 72 hours and measured by flow cytometry. Results are presented as MFI. Mean±SD. n=5. (B) CD14+ monocytes were incubated with HMGB1 or S100A9 for 1 hour in the presence of resatorvid or FPS-ZM1. Expression of p-p38 and p-p65 were measured by western blot. The representative experiment out of three is shown. Quantification of the expression was performed by normalizing the band intensities of phosphorylated protein to total protein. Mean±SD. n=3. Cumulative data for the expression of (C) p-p38 and (D) p-p65 in monocytes treated with HMGB1 or S100A9 for 72 hours and measured by flow cytometry. Data are presented as MFI. Mean±SD. n=5–6. (E) pSTAT3 expression in monocytes treated with HMGB1 or S100A9 for 24 hours determined by western blot. The representative experiment out of three is shown. Quantification of the expression was performed by normalizing the band intensities of phosphorylated STAT3 to total STAT3. Mean±SD. n=3. (F) Cumulative data for pSTA3 expression in monocytes treated with HMGB1 or S100A9 for 72 hours measured by flow cytometry. Data are depicted as MFI. Mean±SD. n=8–10. P value was calculated by a two-sided paired t-test (A, B, E). P value was calculated by Wilcoxon test (C, D, F). *p<0.05, **p<0.01. MFI, mean fluorescence intensity; RAGE, receptor for advanced glycation endproducts; STAT3, signal transducer and activator of transcription factor 3; TLR4, toll like receptor 4.

Fig 3: S100A9 and HMGB1 convert monocytes into immunosuppressive MDSC through TLR4 signaling. Monocytes were incubated for 72 hours with S100A9 or HMGB1 (both 5 µg/mL) in the presence of GM-CSF (40 ng/mL). The immunosuppressive capacity of monocytes was determined by flow cytometry after their co-culture for 96 hours with activated CD3+ T cells labeled with the proliferation dye. (A) Representative histograms and cumulative data for T cells cultured for 96 hours alone (unstim), activated with anti-CD3 and anti-CD28 mAbs (stim), or co-cultured with monocytes treated only with GM-CSF or GM-CSF with S100A9 or HMGB1. Results are presented as the percentage of divided T cells normalized to the respective control of stimulated T cells (stim). Mean±SD; n=7–19. (B) In some experiments, monocytes were also pretreated for 1 hour with the TLR4 inhibitor resatorvid (5 µM) or the receptor for advanced glycation endproducts inhibitor FPS-ZM1 (30 nM). Data are presented as the percentage of divided T cells normalized to the respective control. Mean±SD. n=6–19. (C–F) The expression of markers on monocytes was measured by flow cytometry. (C) Results are presented as the percentage of PD-L1+ monocytes among total monocytes. Mean±SD. n=4–7. (D) Data for ROS production are shown as mean fluorescence intensity (MFI). Mean±SD. n=5–11. (E) Expression of IDO-1 in S100A9-treated or HMGB1-treated monocytes pretreated with resatorvid or FPS-ZM1 was measured by western blot. The representative experiment out of three is shown. Quantification of the expression was performed by normalizing the intensities of IDO expression to that of GAPDH. Mean±SD. n=3. Data for (F) CD86 and (G) HLA-DR expression are shown as MFI. Mean±SD. n=6–9. (H–J) Microarray analysis of monocytes cultured for 72 hours with S100A9 or pretreated with resatorvid or FPS-ZM1. Volcano plots showing the differentially expressed genes for (H) S100A9-treated versus control, (I) S100A9+resatorvid versus S100A9-treated, and (J) S100A9+FPS-ZM1 versus S100A9-treated monocytes. Monocytes incubated with GM-CSF only were shown as a control. The horizontal dashed line indicates the significance threshold (p<0.05). Selected differentially expressed genes are shown as red (upregulated) and green circles (downregulated). Vertical dashed line indicates twofold change. n=4. P value was calculated by Mann-Whitney test (A, B). P value was calculated by Wilcoxon test (C, D, F, G). *p<0.05, **p<0.01, ***p<0.001. GM-CSF, granulocyte-macrophage colony-stimulating factor; IDO, indoleamine 2,3-dioxygenase 1; MDSC, myeloid-derived suppressor cells; PD-L1, programmed death-ligand 1; ROS, reactive oxygen species; TLR4, toll like receptor 4.

Fig 4: Increased plasma levels of S100A8/9 predict poor PFS in patients with melanoma. (A) The Cancer Genome Atlas analysis of MDSC markers in tumor samples from patients with melanoma stratified into top and bottom 25 percentiles based on S100A9 expression. Differentially upregulated genes in tumors with high S100A9 expression as compared with those in tumors with low S100A9 levels are shown. Dashed vertical line indicates the threshold of statistical significance. n=111. (B–E) The concentration of S100A9 in the plasma of patients with melanoma was measured by ELISA (n=40). The expression of PD-L1 on M-MDSC from these patients was determined by flow cytometry (n=33). (B) The percentage of PD-L1+ M-MDSC among total MDSC was plotted against the plasma levels of S100A8/9 (ng/mL) in patients with melanoma at the baseline (n=33). (C) Plasma levels of S100A8/9 before (baseline) and after first ICI administration (on-treatment) are expressed in ng/mL. Mean±SD. n=10. (D) PFS of patients with melanoma with high (>834.9 ng/mL; n=16) and low (<834.9 ng/mL; n=24) plasma levels of S100A8/9 at the baseline are shown as a Kaplan-Meier curve. (E) Plasma concentrations of S100A8/9 in patients with melanoma responding (R, n=17) and non-responding (NR, n=23) to ICI treatment were expressed in ng/mL. Mean±SD. P value was calculated by Pearson correlation with a two-sided p value (B). P value was calculated by Wilcoxon test (C). P value was calculated by logrank (Mantel-Cox) test (D). P value was calculated by Mann-Whitney test (A, E). *p<0.05, **p<0.01. ARG1, arginase-1; ICI, immune checkpoint inhibitors; IL, interleukin; M-MDSC, mononuclear MDSC; MDSC, myeloid-derived suppressor cells; PD-L1, programmed death-ligand 1; PFS, progression-free survival.