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

Zhang M, Hutter G, Kahn SA, Azad TD, Gholamin S, Xu CY, Liu J, Achrol AS, Richard C, Sommerkamp P, Schoen MK, McCracken MN, Majeti R, Weissman I, Mitra SS, Cheshier SH - PLoS ONE (2016)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatric Neurosurgery, Department of Neurosurgery, Lucile Packard Children's Hospital, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

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.(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.
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pone.0153550.g003: 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.(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.

Mentions: To appreciate the effect that Fc-receptor mediated opsonization can have on coinciding Sirp-CD47 disruption on phagocytosis, we performed additional phagocytosis assays with Fc-blocked monoclonal antibodies toward GBM primary lines, thereby reducing nonspecific IgG binding. This strategy of limiting the Fc-receptor contribution continued to yield a phagocytosis rate that was significantly higher than that of IgG4-only control, however it was significantly albeit marginally decreased from that of the unblocked antibody (Fig 3A). To examine how IgG subclass may affect antibody-mediated cellular phagocytosis (ADCP) effects and CD47-Sirp disruption-induced phagocytosis, we treated glioblastoma cells with an IgG1-antibody against an unrelated target, epidermal growth factor receptor (EGFR, cetuximab). Unlike after Fc-receptor blockade of Hu5F9-G4, where a significant contribution of CD47-Sirp disruption towards the overall phagocytosis persisted, the ADCP-effect of Cetuximab significantly decreased upon blocking Fc receptors on macrophages. To detect the baseline antibody-mediated cellular cytotoxicity, we performed an additional comparison of tumor cells (GBM6) and macrophages (M0) pretreated with anti-CD47 or isotype-matched antibody (mouse IgG4) before coincubation. Only pretreating tumor cells with Hu5F9-G4, and not the macrophages, resulted in an increase of phagocytosis (Fig 3B). Pretreatment of macrophages and subsequent wash-out of the antibody did not influence tumor cells phagocytosis (Fig 3B).


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

Zhang M, Hutter G, Kahn SA, Azad TD, Gholamin S, Xu CY, Liu J, Achrol AS, Richard C, Sommerkamp P, Schoen MK, McCracken MN, Majeti R, Weissman I, Mitra SS, Cheshier SH - PLoS ONE (2016)

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.(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.
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Related In: Results  -  Collection

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pone.0153550.g003: 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.(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.
Mentions: To appreciate the effect that Fc-receptor mediated opsonization can have on coinciding Sirp-CD47 disruption on phagocytosis, we performed additional phagocytosis assays with Fc-blocked monoclonal antibodies toward GBM primary lines, thereby reducing nonspecific IgG binding. This strategy of limiting the Fc-receptor contribution continued to yield a phagocytosis rate that was significantly higher than that of IgG4-only control, however it was significantly albeit marginally decreased from that of the unblocked antibody (Fig 3A). To examine how IgG subclass may affect antibody-mediated cellular phagocytosis (ADCP) effects and CD47-Sirp disruption-induced phagocytosis, we treated glioblastoma cells with an IgG1-antibody against an unrelated target, epidermal growth factor receptor (EGFR, cetuximab). Unlike after Fc-receptor blockade of Hu5F9-G4, where a significant contribution of CD47-Sirp disruption towards the overall phagocytosis persisted, the ADCP-effect of Cetuximab significantly decreased upon blocking Fc receptors on macrophages. To detect the baseline antibody-mediated cellular cytotoxicity, we performed an additional comparison of tumor cells (GBM6) and macrophages (M0) pretreated with anti-CD47 or isotype-matched antibody (mouse IgG4) before coincubation. Only pretreating tumor cells with Hu5F9-G4, and not the macrophages, resulted in an increase of phagocytosis (Fig 3B). Pretreatment of macrophages and subsequent wash-out of the antibody did not influence tumor cells phagocytosis (Fig 3B).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatric Neurosurgery, Department of Neurosurgery, Lucile Packard Children's Hospital, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus