Fig. 2

Activation of osteoblasts via PTH1R ligands (PTH or PTHrP) releases M-MDSCs binding to osteoblasts. a Schematic representation of the in vitro cell binding assay. Murine or human M-MDSCs were isolated by flow cytometric sorting from 4T1 tumor-bearing mouse tibia or breast cancer patient blood, followed by CFSE labeling and coculture with primary calvarial (murine) or hFOB1.19 (human) osteoblast monolayer cultures, respectively. b, c Microscopic images of murine primary calvarial osteoblasts (b) or human hFOB1.19 (c) osteoblasts (Vibrant® DiD-NIR dye; orange) cocultured with murine or human M-MDSCs (CFSE; green), respectively, for 15 min. d, e Addition of rhPTHrP(1-34) or rhPTH(1-34), both functional ligands for the common receptor PTH1R, released M-MDSC binding to osteoblasts. f Effects of rhPTHrP (1-34, 1 or 10 nmol·L⁻¹) or rhPTH (1-34, 1 or 10 nmol·L⁻¹) on M-MDSC and osteoblast binding release were inhibited by SQ22,356 (SQ, 0, 10 or 100 μmol·L⁻¹), an inhibitor of adenylate cyclase downstream of PTH1R. g Semiquantitative RT‒PCR showing PTH1R expression in murine (primary calvarial or MC3T3E1 cell line) and human hFOB1.19 osteoblasts but not in M-MDSCs or breast tumor cells (4T1 murine or MCF7 and MDA-MB-231 human cell lines). h–j PTH1R knockdown blunted the effects of PTHrP (1-34, 1 or 10 nmol·L⁻¹) or PTH (1-34, 1 or 10 nmol·L⁻¹) on MDSC-osteoblast binding release. h Western blotting analysis showing decreased expression of PTH1R in murine or human osteoblasts transduced with PTH1R-specific shRNA expression lentiviral vectors. Untreated or scrambled sequence expression vector-transfected cells were used as controls. i, j In vitro cell binding assay using PTH1R knockdown osteoblasts. Data are the mean ± SEM (n = 3 per group). **P < 0.01; ns nonsignificant. One-way ANOVA with multiple group comparisons