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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

Differential phagocytosis rate of human M1 and M2 macrophages towards various human glioma cells upon CD47-SIRPα disruption.(A) Representative flow cytometric phagocytosis assay of human M1 macrophages against CSFE-labeled GBM1 and PGBM1 cells. The percentage of CSFE+CD11b+CD80high 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+CD163high 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).
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pone.0153550.g002: Differential phagocytosis rate of human M1 and M2 macrophages towards various human glioma cells upon CD47-SIRPα disruption.(A) Representative flow cytometric phagocytosis assay of human M1 macrophages against CSFE-labeled GBM1 and PGBM1 cells. The percentage of CSFE+CD11b+CD80high 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+CD163high 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).

Mentions: We used the same in vitro assay to evaluate whether human M1 and M2 macrophage phagocytosis activities differed toward primary GBM lines. Five different primary tumor cell lines, GBM1-4 and PGBM1, were offered to either human M1 or M2 macrophages as targets (Fig 2, S5B Fig). As before, to evaluate the responsiveness of these M1 and M2 macrophages to anti-CD47 treatment, we administered anti-CD47 antibodies to co-cultures. Both M1 and M2 macrophages showed increased phagocytosis of all tumor lines evaluated (Fig 2). The mean phagocytosis rate of human M1 macrophages toward all tumor cells increased significantly upon anti-CD47 treatment (overall increase 47%, p = 0.015, paired t-test, Fig 2A and 2C, S5B Fig). A significant increase of mean phagocytic activity toward almost all tumor cells could also be observed in M2 macrophages (overall increase 24%, p = 0.015, paired t-test, Fig 2B and 2D, S5B Fig). The resulting human M1 macrophage phagocytosis rates after anti-CD47 treatment were significantly higher than those observed in human M2 macrophages in 3 out of 5 tumors (Table 3). However, in GBM2 and GBM3 where we did not see a statistically significant increase in M1 macrophage phagocytosis, we consistently observed a higher phagocytosis rate in M1 macrophages.


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)

Differential phagocytosis rate of human M1 and M2 macrophages towards various human glioma cells upon CD47-SIRPα disruption.(A) Representative flow cytometric phagocytosis assay of human M1 macrophages against CSFE-labeled GBM1 and PGBM1 cells. The percentage of CSFE+CD11b+CD80high 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+CD163high 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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4836698&req=5

pone.0153550.g002: Differential phagocytosis rate of human M1 and M2 macrophages towards various human glioma cells upon CD47-SIRPα disruption.(A) Representative flow cytometric phagocytosis assay of human M1 macrophages against CSFE-labeled GBM1 and PGBM1 cells. The percentage of CSFE+CD11b+CD80high 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+CD163high 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).
Mentions: We used the same in vitro assay to evaluate whether human M1 and M2 macrophage phagocytosis activities differed toward primary GBM lines. Five different primary tumor cell lines, GBM1-4 and PGBM1, were offered to either human M1 or M2 macrophages as targets (Fig 2, S5B Fig). As before, to evaluate the responsiveness of these M1 and M2 macrophages to anti-CD47 treatment, we administered anti-CD47 antibodies to co-cultures. Both M1 and M2 macrophages showed increased phagocytosis of all tumor lines evaluated (Fig 2). The mean phagocytosis rate of human M1 macrophages toward all tumor cells increased significantly upon anti-CD47 treatment (overall increase 47%, p = 0.015, paired t-test, Fig 2A and 2C, S5B Fig). A significant increase of mean phagocytic activity toward almost all tumor cells could also be observed in M2 macrophages (overall increase 24%, p = 0.015, paired t-test, Fig 2B and 2D, S5B Fig). The resulting human M1 macrophage phagocytosis rates after anti-CD47 treatment were significantly higher than those observed in human M2 macrophages in 3 out of 5 tumors (Table 3). However, in GBM2 and GBM3 where we did not see a statistically significant increase in M1 macrophage phagocytosis, we consistently observed a higher phagocytosis rate in M1 macrophages.

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