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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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Morphology of monocyte-derived macrophages after 6 days.Differentiation in the presence of GM-CSF (A) or M-CSF (B) in Xvivo 10 or RPMI +10% FBS. (C) Differentiation in XVivo 10 media supplemented with the indicated cytokines, as described in Material and Methods. The scale denotes 50 µm.
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pone-0042656-g001: Morphology of monocyte-derived macrophages after 6 days.Differentiation in the presence of GM-CSF (A) or M-CSF (B) in Xvivo 10 or RPMI +10% FBS. (C) Differentiation in XVivo 10 media supplemented with the indicated cytokines, as described in Material and Methods. The scale denotes 50 µm.

Mentions: Monocyte-derived macrophages presented a unique morphology dependent on both the culture media and the cytokine stimulation used. Stimulation with GM-CSF in XVivo 10 revealed a different morphology compared to RPMI 10% FBS which was also visible for M-CSF differentiated macrophages in serum-free and serum-containing media. GM-CSF in XVivo 10 or M-CSF in RPMI 10% FBS led to a majority of elongated, fibroblast-like shaped cells whereas the presence of M-CSF in XVivo 10 or GM-CSF in RPMI 10% FBS induced a majority of round or oval macrophages (Figures 1A and 1B). Similar tendency was observed among donors, albeit with variations in the proportion of round versus elongated cells from one donor to the other (data not shown).


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)

Morphology of monocyte-derived macrophages after 6 days.Differentiation in the presence of GM-CSF (A) or M-CSF (B) in Xvivo 10 or RPMI +10% FBS. (C) Differentiation in XVivo 10 media supplemented with the indicated cytokines, as described in Material and Methods. The scale denotes 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042656-g001: Morphology of monocyte-derived macrophages after 6 days.Differentiation in the presence of GM-CSF (A) or M-CSF (B) in Xvivo 10 or RPMI +10% FBS. (C) Differentiation in XVivo 10 media supplemented with the indicated cytokines, as described in Material and Methods. The scale denotes 50 µm.
Mentions: Monocyte-derived macrophages presented a unique morphology dependent on both the culture media and the cytokine stimulation used. Stimulation with GM-CSF in XVivo 10 revealed a different morphology compared to RPMI 10% FBS which was also visible for M-CSF differentiated macrophages in serum-free and serum-containing media. GM-CSF in XVivo 10 or M-CSF in RPMI 10% FBS led to a majority of elongated, fibroblast-like shaped cells whereas the presence of M-CSF in XVivo 10 or GM-CSF in RPMI 10% FBS induced a majority of round or oval macrophages (Figures 1A and 1B). Similar tendency was observed among donors, albeit with variations in the proportion of round versus elongated cells from one donor to the other (data not shown).

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