Anti-CD47 Treatment Stimulates Phagocytosis of Glioblastoma by M1 and M2 Polarized Macrophages and Promotes M1 Polarized Macrophages In Vivo.

Tumor-associated macrophages (TAMs) represent an important cellular subset within the glioblastoma (WHO grade IV) microenvironment and are a potential therapeutic target. TAMs display a continuum of different polarization states between antitumorigenic M1 and protumorigenic M2 phenotypes, with a lower M1/M2 ratio correlating with worse prognosis. Here, we investigated the effect of macrophage polarization on anti-CD47 antibody-mediated phagocytosis of human glioblastoma cells in vitro, as well as the effect of anti-CD47 on the distribution of M1 versus M2 macrophages within human glioblastoma cells grown in mouse xenografts. Bone marrow-derived mouse macrophages and peripheral blood-derived human macrophages were polarized in vitro toward M1 or M2 phenotypes and verified by flow cytometry. Primary human glioblastoma cell lines were offered as targets to mouse and human M1 or M2 polarized macrophages in vitro. The addition of an anti-CD47 monoclonal antibody led to enhanced tumor-cell phagocytosis by mouse and human M1 and M2 macrophages. In both cases, the anti-CD47-induced phagocytosis by M1 was more prominent than that for M2. Dissected tumors from human glioblastoma xenografted within NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and treated with anti-CD47 showed a significant increase of M1 macrophages within the tumor. These data show that anti-CD47 treatment leads to enhanced tumor cell phagocytosis by both M1 and M2 macrophage subtypes with a higher phagocytosis rate by M1 macrophages. Furthermore, these data demonstrate that anti-CD47 treatment alone can shift the phenotype of macrophages toward the M1 subtype in vivo

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

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

CD47 expression of the tested cell lines.

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