Fig. 5

Only the human OS 143B line, when implanted in NSG mice, induces the expansion of murine MDSCs. A NSG mice were engrafted with six different human sarcoma cell lines. Tumor growth was evaluated by In vivo imaging system (IVIS) for five different orthotopically models (200′000 cell/mice) of five OS cell lines (143B, U-2OS, SAOS-2, MG-63, HOS) and one ERMS cell line (RD). No tumor bearing mice have been used as control group. B Weekly plasma samples were collected from each tumor-bearing mouse and control mice, to investigate circulating mMDSCs using FACS flow-cytometry. The analysis looked for both monocytic MDSCs B and polymorphonuclear (PMN) MDSCs (C) cell subset of CD11b + cells. To ensure accurate comparison and data normalization across samples, counting beads were utilized. *p-value = < 0.05; **p-value = < 0.01; ***p-value = < 0.001; ****p-value = < 0.0001, t test. D-E Quantitative analysis of multiple MSDC ligands was performed using the Luminex assay. Supernatant from tumor cultures (143B, MG-63, U-2OS, HOS, SAOS-2 and RD) D and plasma samples of tumor bearing mice (143B, MG-63, U-2OS,, HOS, SAOS-2 and RD) E were explored using a predesigned panel assay, at 72 h of culture and at time of mice sacrifice respectively. Quantification of the specified cytokines, including CCL2, CCL3, CCL5, CCL7, CX3CL1, CXCL1, CXCL2, CXCL4, CXCL5, CXCL6, CXCL8 (IL8), CXCL13, CXCL14, G-CSF (CSF3), GM-CSF, IFN-gamma, IL10, IL13, IL1b, IL5, MCSF, and TNF-alpha, was normalized on blank control background for the in vitro data (in violet) and no tumor sample for the in vivo data (in blu), and values reported in a Heatmap graph. Data are represented as mean ± standard deviation (SD) from n = 3. F The efficiency of CSF3 and CXCL8 KO in 143B cell lines was assessed by ELLA assay on 72 h cell lines supernatants. (G) Comparative Analysis of Crisped 143B OS Cell Lines for CXCL8 and G-CSF3 (CSF3). The cell lines 143B (wild-type control, orange line), 143B CXCL8^−/− (143B CXCL8 knockout, red line), and 143B G-CSF^−/− (143B G-CSF knockout, blue line), where analyzed for GD2 expression (percentage and GD2 MFI) by flow-cytometry and normalized on the Isotype Control. H The 143B wild-type control (dark grey), 143B CXCL8^−/− (white), 143B G-CSF^−/− (light gray) cells were co-cultured for 5 days with NT or GD2.CAR T-cells at E: T = 1:1 ratio. At the endpoint the percentage of residual tumor was evaluated by FACS as measure of GFP tumor expression. Data are expressed as mean ± SD from 3 donor. Statistical significance was determined using ANOVA test with *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001