Differential phagocytosis rate of human M1 and M2 macrophages towards various human glioma cells upon CD47-SIRPα disruption.

<p>(A) Representative flow cytometric phagocytosis assay of human M1 macrophages against CSFE-labeled GBM1 and PGBM1 cells. The percentage of CSFE+CD11b+CD80^high live singlets was measured and compared between untreated (left column) and anti-CD47 antibody-treated (right column) co-cultures. (B) Representative flow cytometric phagocytosis assay of human M2 macrophages against CSFE-labeled GBM1 and PGBM1 cells. The percentage of CSFE+CD11b+CD163^high live singlets was measured and compared between untreated and anti-CD47 antibody-treated co-cultures. (C) Bar graph demonstrating the change in phagocytosis rates by human M1 macrophages towards individual co-incubated (GBM1-4 and PGBM1) tumor cells -/+ anti-CD47 (significant difference in means of technical triplicates indicated by * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, multiple t-tests). (D) Bar graph demonstrating the change in phagocytosis rates by human M2 macrophages towards individual tumor cell (GBM1-4 and PGBM1 -/+ anti-CD47 (significant difference in means of technical triplicates indicated by * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, multiple t-tests).</p

<i>In vivo</i> analysis of tumor-associated macrophage polarization upon anti-CD47 treatment; bioluminescence and survival analysis of treated mice.

<p>(A) Ratio of CD80 and CD206 positive cell count per total macrophage count in untreated and anti-CD47-treated mice (p = 0.0054 for CD80 and 0.164 for CD260, paired t-test). (B) Median fluorescent intensity (MFI) measurement of CD80-AF647 and CD206-PE (p = 0.0002 for CD80 and 0.423 for CD206, paired t-test). (C) Bioluminescence in vivo imaging data (left panel), photon flux values at days 21 and 50 (middle panel) and Kaplan-Meier analysis of mice grafted with GBM5 and treated with Hu5F9-G4 (right panel, 250 μg/dose, every other day, starting at week 3; n = 5 per group, p = 0.0018, log-rank analysis). Legend: blue shapes: mice engrafted with GBM4, black shapes: mice engrafted with GBM5.</p

CD47 expression of the tested cell lines.

<p>CD47 expression of the tested cell lines.</p

Differential phagocytosis rate of mouse M1 and M2 macrophages toward various human glioma cells upon CD47-SIRPα disruption.

<p>(A) Representative flow cytometric phagocytosis assay of mouse M1 macrophages against CSFE-labeled GBM1 and PGBM1 cells. The percentage of CSFE+CD11b+CD80^high live singlets was measured and compared between untreated and anti-CD47 antibody-treated co-cultures. (B) Representative flow cytometric phagocytosis assay of mouse M2 macrophages against CSFE-labeled GBM1 and PGBM1 cells. The percentage of CSFE+CD11b+CD206^high live singlets was measured and compared between untreated (left column) and anti-CD47 antibody-treated (right column) co-cultures. (C) Bar graph demonstrating the change in phagocytosis rates by mouse M1 macrophages towards individual co-incubated cell lines (GBM1, GBM4 and PGBM1) -/+ anti-CD47 (significant difference in means of technical triplicates indicated by * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, multiple t-tests). (D) Bar graph demonstrating the change in phagocytosis rates by mouse M2 macrophages towards individual co-incubated cell lines (GBM1, GBM4 and PGBM1) -/+ anti-CD47 (significant difference in means of technical triplicates indicated by * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, multiple t-tests).</p

M1 and M2 macrophage phagocytosis rates after anti-CD47 treatment.

<p>Data are means of 3 replicates per tumor cell line.</p

Influence of anti-CD47 pretreatment of tumor cells or pretreatment of macrophages on phagocytosis; polarization properties of M1 and M2 macrophages after anti-CD47 treatment in vitro.

<p>(A) Bar graph demonstrating Fc-receptor contribution during anti-CD47 treatment. Anti-CD47 mediated phagocytosis is marginally reduced A monoclonal antibody against EGFR (Cetuximab) was used as an inductor of antibody-dependent cellular phagocytosis (ADCP) which is abrogated upon pre-blocking with an Fc-Blocking peptide. (significant difference in means of technical triplicates indicated by * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, multiple t-tests). (B) Bar graph demonstrating the change in phagocytosis rates by human M0 macrophages towards GBM6 -/+ anti-CD47 pretreatment of tumor cells or pretreatment of macrophages vs. IgG4 isotype-matched control antibody (significant difference in means of technical triplicates indicated by * p ≤ 0.05, multiple t-tests). (C) Overlay contour plots of either resting human M1 or M2 macrophages (CD11b+ CD14+) treated with anti-CD47 antibody or control IgG4 and phagocytosing M1 and M2 macrophages.</p

Cell lines used for <i>in vitro</i> and <i>in vivo</i> experiments, tissue of origin.

<p>Cell lines used for <i>in vitro</i> and <i>in vivo</i> experiments, tissue of origin.</p