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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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Cytokine concentration in supernatant of monocyte-derived macrophages.Monocytes were stimulated for 6 days with (A) GM-CSF (M1) or M-CSF (M2) in XVivo 10 or RPMI +10% FBS, or with (B) GM-CSF for 3 days and LPS and IFN-γ for 3 additional days (M1), M-CSF+IL-4 (M2a) or M-CSF+IL-10 (M2c) in XVivo 10. Data represent mean ± SEM of cytokine concentration of at least 8 donors. Statistical significance was determined using Tukey-Kramer HSD test pairwise comparison (** p<0.005, ***p<0.001).
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pone-0042656-g003: Cytokine concentration in supernatant of monocyte-derived macrophages.Monocytes were stimulated for 6 days with (A) GM-CSF (M1) or M-CSF (M2) in XVivo 10 or RPMI +10% FBS, or with (B) GM-CSF for 3 days and LPS and IFN-γ for 3 additional days (M1), M-CSF+IL-4 (M2a) or M-CSF+IL-10 (M2c) in XVivo 10. Data represent mean ± SEM of cytokine concentration of at least 8 donors. Statistical significance was determined using Tukey-Kramer HSD test pairwise comparison (** p<0.005, ***p<0.001).

Mentions: Levels of 42 cytokines and chemokines in MDM supernatant were determined using the MAPx technology. Direct comparison of cytokine levels between MDM subtypes was conducted owing to a similar number of cells in culture upon differentiation, regardless of the stimulation applied (data not shown). Among the 42 cytokines measured, 30 could be detected above background expression level and 14 showed significant differences in expression levels between M1 and M2 MDM (Figure 3and Table S2A). However, a number of factors were secreted at extremely low level (<50 pg/ml). MDM released high levels of both chemokines CCL2 and IL-8, regardless of the stimulation applied. The levels of cytokine expression were highly variable among donors. However, compiling results from 8 distinct donors showed that levels of CCL3 and TNF-α were higher in M1 compared to M2 supernatant, correlating with the pro-inflammatory nature of these cytokines. On the other hand, the level of IL-10 was higher in M2 supernatant. In both macrophage subpopulations, PDGF-AA and Gro levels were higher in MDM supernatant cultured in XVivo 10 as compared to RPMI. Similarly, IL-1ra and CCL7 secreted by M2 as well as TNF-α, IL-1α, IL-1β, and CCL5 released by M1 were higher in XVivo 10 than in RPMI. In summary, cytokine levels detected in M1 or M2 CM appeared to be higher in serum-free compared to serum-containing media.


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)

Cytokine concentration in supernatant of monocyte-derived macrophages.Monocytes were stimulated for 6 days with (A) GM-CSF (M1) or M-CSF (M2) in XVivo 10 or RPMI +10% FBS, or with (B) GM-CSF for 3 days and LPS and IFN-γ for 3 additional days (M1), M-CSF+IL-4 (M2a) or M-CSF+IL-10 (M2c) in XVivo 10. Data represent mean ± SEM of cytokine concentration of at least 8 donors. Statistical significance was determined using Tukey-Kramer HSD test pairwise comparison (** p<0.005, ***p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412794&req=5

pone-0042656-g003: Cytokine concentration in supernatant of monocyte-derived macrophages.Monocytes were stimulated for 6 days with (A) GM-CSF (M1) or M-CSF (M2) in XVivo 10 or RPMI +10% FBS, or with (B) GM-CSF for 3 days and LPS and IFN-γ for 3 additional days (M1), M-CSF+IL-4 (M2a) or M-CSF+IL-10 (M2c) in XVivo 10. Data represent mean ± SEM of cytokine concentration of at least 8 donors. Statistical significance was determined using Tukey-Kramer HSD test pairwise comparison (** p<0.005, ***p<0.001).
Mentions: Levels of 42 cytokines and chemokines in MDM supernatant were determined using the MAPx technology. Direct comparison of cytokine levels between MDM subtypes was conducted owing to a similar number of cells in culture upon differentiation, regardless of the stimulation applied (data not shown). Among the 42 cytokines measured, 30 could be detected above background expression level and 14 showed significant differences in expression levels between M1 and M2 MDM (Figure 3and Table S2A). However, a number of factors were secreted at extremely low level (<50 pg/ml). MDM released high levels of both chemokines CCL2 and IL-8, regardless of the stimulation applied. The levels of cytokine expression were highly variable among donors. However, compiling results from 8 distinct donors showed that levels of CCL3 and TNF-α were higher in M1 compared to M2 supernatant, correlating with the pro-inflammatory nature of these cytokines. On the other hand, the level of IL-10 was higher in M2 supernatant. In both macrophage subpopulations, PDGF-AA and Gro levels were higher in MDM supernatant cultured in XVivo 10 as compared to RPMI. Similarly, IL-1ra and CCL7 secreted by M2 as well as TNF-α, IL-1α, IL-1β, and CCL5 released by M1 were higher in XVivo 10 than in RPMI. In summary, cytokine levels detected in M1 or M2 CM appeared to be higher in serum-free compared to serum-containing media.

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