Next, we investigated the effects of cyclophosphamide treatment on the MM cell secretome and subsequent immune effector (macrophage) cell recruitment and functional responses. the migratory capacity of macrophages and increased CD32 and CD64 Fc receptor expression on their cell surface. Daratumumab-specific tumor clearance was increased by conditioning macrophages with CTX-TCS in a dose-dependent manner. This effect was impeded by pre-incubating macrophages with Cytochalasin D (CytoD), an inhibitor of actin polymerization, indicating macrophage-mediated ADCP as the mechanism of clearance. CD64 expression P2RY5 on macrophages directly correlated with MM cell clearance and was essential to the observed synergy between cyclophosphamide and daratumumab, as tumor clearance was attenuated in the presence of a FcRI/CD64 blocking agent. Cyclophosphamide independently enhances daratumumab-mediated killing of MM cells by altering the tumor microenvironment to promote macrophage recruitment, polarization to a pro-inflammatory phenotype, and directing ADCP. These findings support the addition of cyclophosphamide to existing or novel monoclonal antibody-containing MM regimens. efficacy Cisatracurium besylate of antibodies and improved outcome has been seen in conjunction with high affinity polymorphisms of FcRIIa/CD32a, which are not expressed on NK cells.27 This may be particularly important in the context of treatment with daratumumab. Originally, it was thought that ADCC mediated by NK cells would constitute one of the most important mechanisms of action of daratumumab.28 However, with the benefit of careful correlative studies from clinical trials, we now know that treatment with daratumumab leads to rapid depletion of NK cells, which are strongly CD38 positive, and that this can last up to 6?months following cessation of treatment.29 Therefore, to maximize Cisatracurium besylate the clinical efficacy of daratumumab, it may be necessary to have a sufficient number of activated TAMs. 30 This has recently been demonstrated by Viola data from a clinical trial, set up to assess the addition of cyclophosphamide to a daratumumab-containing regimen (CyBorD-DARA), uncovered observations suggesting that this treatment combination increased the vulnerability of MM cells to phagocytosis by macrophages.30 Our study investigates the specific mechanism of action of cyclophosphamide in the induction of ADCP test. *0.05. **0.01. ***0.001 Macrophage migration assay THP-1 cells were serum-starved for 24?hours. THP-1 cell suspensions (1x105 cells) were added into the top wells of 8?m transwell inserts which were placed into a 24-well plate. Culture media was placed into Cisatracurium besylate the bottom of the 24-well plate (as depicted in the Figure 2(e) schematic). 10% FBS-containing culture media and 20% FBS-containing culture media were used as positive migration controls. TCS released from cyclophosphamide treated MM cells (CTX-TCS), as described above, was placed into the wells of the 24-well plate in the absence or presence of 0.1?g/ml anti-CCL5 (R&D Systems, MN, USA). The plate was returned to the incubator at 37C for 4?hours, after which the media containing migrated THP-1 cells in the 24-well plates was collected. The media was centrifuged (400?g for 5?minutes), the cells were collected and counted using an Accuri? C6 flow cytometer (BD Biosciences). Cell number was reported as absolute counts. Human peripheral blood mononuclear cell isolation Freshly drawn peripheral blood (PB) was collected in 10?ml ethylene diamine tetraacetic acid (EDTA) Vacutainer? tubes (BD Medical Supplies, Crawley, UK). Mononuclear cells (MC) were isolated from PB in 15?ml falcon tubes (Sarstedt, Wexford, Ireland) by layering 3?ml EDTA-anti-coagulated sample over 3?ml endotoxin-free Ficoll?-Paque density-gradient medium (GE Healthcare, Little Chalfont, United Kingdom). Samples were centrifuged for 22?minutes at 420?g at 4C without brake. Using a plastic Pasteur pipette (Sarstedt), the visible buffy coat layer of mononuclear cells was removed. PBMCs were transferred into a fresh falcon tube (Sarsedt) and subsequently washed with 10?ml fluorescence-activated cell sorting (FACS buffer) [phosphate buffered saline (Thermofisher Scientific), 2% FBS (Sigma), and 0.05% sodium azide (Sigma)]. This suspension was centrifuged Cisatracurium besylate at 300?g for 10?minutes at 4C. The pelleted mononuclear cells were re-suspended in 1?ml FACS buffer after the supernatant was discarded. Isolated PBMCs were treated with the reduced form of cyclophosphamide (CTX) (Santa Cruz Biotechnology) (0?M, 2.5?M, 5?M, 10?M and 20?M) for 24?hours and subsequently stained for phenotypic characterization by flow cytometry. This research was approved by the local hospital ethics Cisatracurium besylate committee according to the requirements.