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In vitro generation of monocyte-derived macrophages under serum-free conditions improves their tumor promoting functions.

Rey-Giraud F, Hafner M, Ries CH - PLoS ONE (2012)

Bottom Line: In this study, we compared the properties of macrophages originating from monocytes cultured either in media containing serum together with M-CSF for M2 and GM-CSF for M1 macrophages or in serum-free media supplemented with M-CSF or GM-CSF and cytokines such as IL-4, IL-10 to induce activated M2 or LPS together with IFN-γ to generate activated M1 phenotype.We observed differences in cell morphology as well as increased surface receptor expression levels in serum-containing culture whereas similar or higher cytokine production levels were detected under serum-free culture conditions.Moreover, evaluation of MDM phagocytic activity in serum free condition resulted in greater phagocytic properties of M2 compared to M1.

View Article: PubMed Central - PubMed

Affiliation: Pharma Research and Early Development, Roche Diagnostics GmbH, Penzberg, Germany.

ABSTRACT
The tumor promoting role of M2 macrophages has been described in in vivo models and the presence of macrophages in certain tumor types has been linked to a poor clinical outcome. In light of burgeoning activities to clinically develop new therapies targeting tumor-associated macrophages (TAMs), reliable in vitro models faithfully mimicking the tumor promoting functions of TAMs are required. Generation and activation of human monocyte-derived macrophages (MDM) in vitro, described as M1 or M2 macrophages attributed with tumoricidal or tumor-promoting functions, respectively, has been widely reported using mainly serum containing culture methods. In this study, we compared the properties of macrophages originating from monocytes cultured either in media containing serum together with M-CSF for M2 and GM-CSF for M1 macrophages or in serum-free media supplemented with M-CSF or GM-CSF and cytokines such as IL-4, IL-10 to induce activated M2 or LPS together with IFN-γ to generate activated M1 phenotype. We observed differences in cell morphology as well as increased surface receptor expression levels in serum-containing culture whereas similar or higher cytokine production levels were detected under serum-free culture conditions. More importantly, MDM differentiated under serum-free conditions displayed enhanced tumoricidal activity for M1 and tumor promoting property for M2 macrophages in contrast to MDM differentiated in the presence of serum. Moreover, evaluation of MDM phagocytic activity in serum free condition resulted in greater phagocytic properties of M2 compared to M1. Our data therefore confirm the tumor promoting properties of M2 macrophages in vitro and encourage the targeting of TAMs for cancer therapy.

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Receptor expression level on monocyte-derived macrophages.Surface expression of CD163 (A) or CD206 (B) on M1 and M2 MDM in Xvivo 10 or RPMI +10% FBS, or on M1, M2a and M2c MDM in XVivo 10 media. Data represent mean ± SEM of Mean Fluorescence Intensity (Geom. mean) of at least 8 donors. Statistical significance was determined using Tukey-Kramer HSD test pairwise comparison (***p<0.001).
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pone-0042656-g002: Receptor expression level on monocyte-derived macrophages.Surface expression of CD163 (A) or CD206 (B) on M1 and M2 MDM in Xvivo 10 or RPMI +10% FBS, or on M1, M2a and M2c MDM in XVivo 10 media. Data represent mean ± SEM of Mean Fluorescence Intensity (Geom. mean) of at least 8 donors. Statistical significance was determined using Tukey-Kramer HSD test pairwise comparison (***p<0.001).

Mentions: When compared to expression level on monocytes, levels of CD45, CD68, CD71, CD64 (FcγRI), HLA-ABC (MHC class I) and HLA-DR (MHC class II), CD86 and CD206 (mannose receptor) were increased during differentiation into macrophages, independently of the culture conditions or macrophage subtype. Moreover, M1 and M2 shared similar expression level of MHC class I, FcγRI and CD86 receptors as well as intracellular CD68 (Table S1A). As previously reported, M1 were characterized by the expression of CD80 and the absence of CD163 (scavenger receptor, Figure 2A) compared to monocytes whereas M2 macrophages expressed higher level of CD14, CD163, CD32 (FcγRII) and CD16 (FcγRIII) than M1 macrophages and lack CD80 expression [4].


In vitro generation of monocyte-derived macrophages under serum-free conditions improves their tumor promoting functions.

Rey-Giraud F, Hafner M, Ries CH - PLoS ONE (2012)

Receptor expression level on monocyte-derived macrophages.Surface expression of CD163 (A) or CD206 (B) on M1 and M2 MDM in Xvivo 10 or RPMI +10% FBS, or on M1, M2a and M2c MDM in XVivo 10 media. Data represent mean ± SEM of Mean Fluorescence Intensity (Geom. mean) of at least 8 donors. Statistical significance was determined using Tukey-Kramer HSD test pairwise comparison (***p<0.001).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412794&req=5

pone-0042656-g002: Receptor expression level on monocyte-derived macrophages.Surface expression of CD163 (A) or CD206 (B) on M1 and M2 MDM in Xvivo 10 or RPMI +10% FBS, or on M1, M2a and M2c MDM in XVivo 10 media. Data represent mean ± SEM of Mean Fluorescence Intensity (Geom. mean) of at least 8 donors. Statistical significance was determined using Tukey-Kramer HSD test pairwise comparison (***p<0.001).
Mentions: When compared to expression level on monocytes, levels of CD45, CD68, CD71, CD64 (FcγRI), HLA-ABC (MHC class I) and HLA-DR (MHC class II), CD86 and CD206 (mannose receptor) were increased during differentiation into macrophages, independently of the culture conditions or macrophage subtype. Moreover, M1 and M2 shared similar expression level of MHC class I, FcγRI and CD86 receptors as well as intracellular CD68 (Table S1A). As previously reported, M1 were characterized by the expression of CD80 and the absence of CD163 (scavenger receptor, Figure 2A) compared to monocytes whereas M2 macrophages expressed higher level of CD14, CD163, CD32 (FcγRII) and CD16 (FcγRIII) than M1 macrophages and lack CD80 expression [4].

Bottom Line: In this study, we compared the properties of macrophages originating from monocytes cultured either in media containing serum together with M-CSF for M2 and GM-CSF for M1 macrophages or in serum-free media supplemented with M-CSF or GM-CSF and cytokines such as IL-4, IL-10 to induce activated M2 or LPS together with IFN-γ to generate activated M1 phenotype.We observed differences in cell morphology as well as increased surface receptor expression levels in serum-containing culture whereas similar or higher cytokine production levels were detected under serum-free culture conditions.Moreover, evaluation of MDM phagocytic activity in serum free condition resulted in greater phagocytic properties of M2 compared to M1.

View Article: PubMed Central - PubMed

Affiliation: Pharma Research and Early Development, Roche Diagnostics GmbH, Penzberg, Germany.

ABSTRACT
The tumor promoting role of M2 macrophages has been described in in vivo models and the presence of macrophages in certain tumor types has been linked to a poor clinical outcome. In light of burgeoning activities to clinically develop new therapies targeting tumor-associated macrophages (TAMs), reliable in vitro models faithfully mimicking the tumor promoting functions of TAMs are required. Generation and activation of human monocyte-derived macrophages (MDM) in vitro, described as M1 or M2 macrophages attributed with tumoricidal or tumor-promoting functions, respectively, has been widely reported using mainly serum containing culture methods. In this study, we compared the properties of macrophages originating from monocytes cultured either in media containing serum together with M-CSF for M2 and GM-CSF for M1 macrophages or in serum-free media supplemented with M-CSF or GM-CSF and cytokines such as IL-4, IL-10 to induce activated M2 or LPS together with IFN-γ to generate activated M1 phenotype. We observed differences in cell morphology as well as increased surface receptor expression levels in serum-containing culture whereas similar or higher cytokine production levels were detected under serum-free culture conditions. More importantly, MDM differentiated under serum-free conditions displayed enhanced tumoricidal activity for M1 and tumor promoting property for M2 macrophages in contrast to MDM differentiated in the presence of serum. Moreover, evaluation of MDM phagocytic activity in serum free condition resulted in greater phagocytic properties of M2 compared to M1. Our data therefore confirm the tumor promoting properties of M2 macrophages in vitro and encourage the targeting of TAMs for cancer therapy.

Show MeSH
Related in: MedlinePlus