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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

In vivo analysis of tumor-associated macrophage polarization upon anti-CD47 treatment; bioluminescence and survival analysis of treated mice.(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.
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pone.0153550.g004: In vivo analysis of tumor-associated macrophage polarization upon anti-CD47 treatment; bioluminescence and survival analysis of treated mice.(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.

Mentions: Last, we investigated whether anti-CD47 treatment in vivo changed the macrophage polarization profile toward a favorable anti-tumorigenic microenvironment. Mice that had been orthotopically xenografted with primary brain tumor cells from a low-passage primary glioma line (GBM5) or a different, highly aggressive glioma cell line (GBM4), and confirmed by bioluminescence imaging, were subsequently treated with either anti-CD47 antibody or control for 4 weeks (1 week in case of engrafted GBM4) after tumor induction. The resulting tumor-bearing brain tissue was analyzed by FACS for its mouse macrophage surface marker profile. Mice treated with anti-CD47 monoclonal antibodies were found to have an increased number of macrophages with M1 phenotype markers relative to untreated mice (data not shown). The ratio of M1 cell counts (CD80+/total macrophage number) was significantly higher in treated mice (Fig 4A). Further, median fluorescence intensity values of the M1 marker CD80-AF647 within the macrophage gate were significantly elevated in anti-CD47-treated mice (Fig 4B). M2 macrophages, as measured by CD206 fluorescence intensity ratios and cell count normalized to total macrophage count, increased within the tumor as well, but to a much lesser extent (Fig 4B). Further, anti-CD47 treatment was effective in reducing tumor burden of GBM5 xenografted mice as measured by bioluminescence and resulted in a significant survival benefit (Fig 4C).


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)

In vivo analysis of tumor-associated macrophage polarization upon anti-CD47 treatment; bioluminescence and survival analysis of treated mice.(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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4836698&req=5

pone.0153550.g004: In vivo analysis of tumor-associated macrophage polarization upon anti-CD47 treatment; bioluminescence and survival analysis of treated mice.(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.
Mentions: Last, we investigated whether anti-CD47 treatment in vivo changed the macrophage polarization profile toward a favorable anti-tumorigenic microenvironment. Mice that had been orthotopically xenografted with primary brain tumor cells from a low-passage primary glioma line (GBM5) or a different, highly aggressive glioma cell line (GBM4), and confirmed by bioluminescence imaging, were subsequently treated with either anti-CD47 antibody or control for 4 weeks (1 week in case of engrafted GBM4) after tumor induction. The resulting tumor-bearing brain tissue was analyzed by FACS for its mouse macrophage surface marker profile. Mice treated with anti-CD47 monoclonal antibodies were found to have an increased number of macrophages with M1 phenotype markers relative to untreated mice (data not shown). The ratio of M1 cell counts (CD80+/total macrophage number) was significantly higher in treated mice (Fig 4A). Further, median fluorescence intensity values of the M1 marker CD80-AF647 within the macrophage gate were significantly elevated in anti-CD47-treated mice (Fig 4B). M2 macrophages, as measured by CD206 fluorescence intensity ratios and cell count normalized to total macrophage count, increased within the tumor as well, but to a much lesser extent (Fig 4B). Further, anti-CD47 treatment was effective in reducing tumor burden of GBM5 xenografted mice as measured by bioluminescence and resulted in a significant survival benefit (Fig 4C).

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