Fig 2: Characterization of markers for ES-phagocytic macrophages. A Gating strategy for evaluating cell surface protein expression on phagocytic versus non-phagocytic macrophages. In-vitro phagocytosis assay was carried out incubating A673 cells (Non- or DOX- or MAG- or D&M-treated) labeled with CMFDA (detected by FITC channel) and M-CSF Mφ. Cells were harvested and stained with APC-CD11b and PE-CD38 antibodies followed by flow cytometry analysis. The viable cells were plotted in FITC (CMFDA) x APC view to visualize cell populations in the in-vitro phagocytosis assay. A gate was drawn to include cells that were FITC positive and APC positive as the phagocytic macrophage population, and another gate was drawn to include cells that were FITC- APC + as the non-phagocytic macrophage population. The FITC + APC + and FITC- APC + cells were then plotted separately in a PE x APC view to visualize expression of CD38 in both cell populations. A quat gate was drawn to visualize APC + PE + as the CD38 + and APC + PE- as the CD38- macrophages. B Comparison of percent of CD14, CD38, CD163, CD206 or CD209 positive and negative cells in the phagocytic and non-phagocytic macrophages incubated with non- or DOX- or MAG- or D&M-treated A673 cells. Macrophages alone (no A673) served as a control. *p < 0.05, **p < 0.01, ***p < 0.001 (Fisher’s Exact test). Shown are results in a representative experiment. The same trend was seen in three independent biological repeats

Fig 3: Effects of NKTR-255 on NK/CAR-NK cells and magrolimab (MAG) on macrophage phagocytosis of Ewing sarcoma (ES) cells in vitro. (A) Effects of NKTR-255 on the expression of NK cell receptors. Ex vivo expanded natural killer (NK) cells were incubated with NKTR-255 (40 ng/mL) for various periods of time (0–6 days) in the absence of IL-2 with replenishment of media and NKTR-255 at day 3. Flow cytometry was performed to detect the expression of NK cell receptors (CD94, NKp30, NKG2D, NKG2A, KIR and NKp44). Representative results from 4 days of incubation were shown. The same trend was seen in three independent biological repeats. (B) Effects of NKTR-255 on NK cell survival and expansion in the absence of IL-2 in vitro. NK cells (2.5×106) at 14 days of expansion were incubated with NKTR-255 (40 ng/mL) for various periods of time (0–6 days) in the absence of IL-2 with replenishment of media and NKTR-255 at day 3. Viable cells were counted by the method of trypan blue staining. Error bars indicate the SD of triplicate samples in a representative experiment. The same trend was seen in three independent biological repeats. *p<0.05, **p<0.01 (two-tailed Student’s t-test). (C) In vitro cytotoxic activity of anti-MCAM-CAR-NK cells in combination with NKTR-255 against ES cells. CAR-NK cells were incubated with or without NKTR-255 (40 ng/mL) in the absence of IL-2 for 3 days before incubating with luciferase-expressing ES cells (A673, TC32, SKNMC) at E:T ratio of 0.2:1 for 4 hours. Cytotoxicity was analyzed by addition of D-luciferin and bioluminescent was measured with a luminometer. *p<0.05, **p<0.01 (two-tailed Student’s t-test). Error bars indicate the SD of triplicate samples in a representative experiment. The same trend was seen in three independent biological repeats. (D) CD47 expression on ES cells. CD47 expression levels were detected by flow cytometry on ES A673, TC32, SKNMC cells. (E) CD47 expression on ES PDX tumors. Immunofluorescent staining by CD47 antibody was performed to detect CD47 (green) expression on MSK-3 and NCH-EW-1 ES PDX tumors. The nuclei were counterstained with DAPI (blue). (F) MAG significantly enhanced macrophage phagocytosis of ES cells. Macrophages were derived from healthy donor peripheral blood mononuclear cells by recombinant human M-CSF (100 ng/mL). ES A673, TC32, SKNMC cells were labeled with CMFDA dye (recognized by FITC channel) and co-cultured with macrophages at a 2:1 ratio together with IgG or MAG (1 µg/mL) for 4 hours. Cells were harvested and stained with α-CD11b followed by flow cytometry analysis. The gated population (CD11b+FITC+) in the dot plots (left) show macrophages phagocytosed ES cells. Right, significantly enhanced macrophage phagocytosis of ES cells (A673, TC32, EWS502) by MAG (blue) compared with IgG control (orange). Quantification of the results in three biological repeats is shown. *p<0.05, **p<0.01 (two-tailed Student’s t-test). E:T, effector-to-target; PDX, patient-derived xenograft.

Fig 4: CD47 blockade magrolimab (MAG) enhanced macrophage phagocytosis of ES cells in vitro and had limited anti-tumor effect in vivo against ES. A Phenotypes of macrophages derived from peripheral blood monocytes by GM-CSF or M-CSF induction. The peaks in pink represent cells stained with isotypes and the ones in blue are cells stained with antibodies against the indicated surface proteins (CD11b, CD14, CD80, CD68, CD163). B Immunofluorescent-based phagocytosis assays showing the increased phagocytosis of A673 cells by both GM-CSF-Mφ and M-CSF-Mφ upon MAG treatment compared to IgG control. A673 cells were labeled with the CMFDA green cell tracker and macrophages the CMAC blue cell tracker. The white arrows are pointing to the macrophages phagocytosed A673 cells. C Quantification of the number of macrophages phagocytosed tumor cells in B. Average number of cells in five fields is shown. Error bars represent the standard deviation (STD). D MAG treatment enhanced phagocytosis of A673 cells by GM-CSF-Mφ or M-CSF-Mφ compared to IgG control in flow cytometry-based phagocytosis assays. A673 cells were labeled with CMFDA which can be detected by FITC channel. Macrophages were stained with CD11b antibody. The gated FITC + CD11b + cells are macrophages phagocytosed A673 cells. The bar graph shows quantification of the results in three independent biological replicates. E Flow cytometry analysis showing CD47 knockout (KO) by the CRISPR/Cas9 approach. F Flow cytometry results showing MAG treatment failed to increase macrophage phagocytosis of CD47 KO A673 cells. Quantification of the results in three biological repeat experiments is shown. **p < 0.01; ns, not significant (two-tailed Student t-test). G MAG treatment significantly enhanced in-vitro phagocytosis of ES A673, EWS502 and TC32 cells. *p < 0.05, **p < 0.01 (two-tailed Student t-test). H Schematic representation of the animal work schedule. Luciferase expressing A673 cells were injected subcutaneously into the flanks of NSG mice. Tumor bearing mice were divided into two groups and treated with IgG and MAG (150 μg/animal, once per day for 12 days, intraperitoneal), respectively. Tumor growth was monitored by weekly IVIS imaging. I Tumor growth curves showing significant effects of MAG on primary tumor growth. N = 10 per group. **p < 0.01 (ANOVA). J Kaplan–Meier curves for comparison of survival between IgG and MAG groups. Animal survival was followed after therapy initiation using death or sacrifice as the terminal event. IgG vs MAG ***p < 0.001 (log rank test)

Fig 5: Platelet and monocyte activation markers in MPAhi versus MPAlo groups.Flow cytometry of whole blood was performed to analyze platelet and monocyte activation markers. (A–D) Expression of platelet activation markers PAC-1, P-selectin, CD40L, and CD40 were compared between MPAhi (n = 32) and MPAlo (n = 28) groups in the resting state and in the presence of the agonists epinephrine (epi), thrombin (thr), adenosine diphosphate (ADP), and arachidonic acid (AA). (E) Spearman’s correlation of MPA levels, platelet activation markers in the resting state measured by flow cytometry, and aggregation in response to different agonists measured by light transmission aggregometry (LTA) is shown. (F) Monocyte activation markers CD11b, CD86, and CD40 were measured using flow cytometry. Graphs show median and interquartile range, Man-Whitney U test was applied. *P < 0.05, **P < 0.01, ***P < 0.001.