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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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Effect of monocyte-derived macrophage supernatant on HCC1143 cell line proliferation.Evaluation was performed in the presence or the absence of neutralizing TNF-α antibodies, in (A) conditioned media from M1 or M2 MDM cultured in RPMI 10% FBS or XVivo 10, (B) conditioned media from activated M1 (act. M1), M2a and M2c MDM stimulated in XVivo 10. Identical y-axis scales were used for comparison sake. Data represent mean ± SEM of three independent experiments including each condition in triplicate. Statistical significance was determined using t-test pairwise comparison (** p<0.005).
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pone-0042656-g004: Effect of monocyte-derived macrophage supernatant on HCC1143 cell line proliferation.Evaluation was performed in the presence or the absence of neutralizing TNF-α antibodies, in (A) conditioned media from M1 or M2 MDM cultured in RPMI 10% FBS or XVivo 10, (B) conditioned media from activated M1 (act. M1), M2a and M2c MDM stimulated in XVivo 10. Identical y-axis scales were used for comparison sake. Data represent mean ± SEM of three independent experiments including each condition in triplicate. Statistical significance was determined using t-test pairwise comparison (** p<0.005).

Mentions: Both M1 and M2 conditioned media from culture in RPMI 10% FBS induced tumor cell growth inhibition at a similar level. However, different results were obtained when using CM from XVivo 10 culture. Indeed, M1 CM slightly reduced cell proliferation whereas M2 CM increased HCC1143 proliferation by 25% (Figure 4A). Decrease in cell proliferation was TNF-α independent as shown by the absence of effect upon addition of a neutralizing TNF-α antibody to the culture. We then assessed the effect of CM from activated M1, M2a and M2c culture on the tumor line viability (Figure 4B). HCC1143 proliferation was not affected in the presence of M2a CM whereas M2c increased cell growth by 25%. In activated M1 CM, HCC1143 proliferation was reduced by 60%. Inhibition of proliferation could be partially rescued by a neutralizing TNF-α antibody. IFN-γ and TGF-β neutralizing antibodies had no effect on tumor cell inhibition (data not shown). The data presented here were also confirmed using T47D and other tumor cell lines (Figure S1 and data not shown). Our data not only revealed significant differences in tumor cell line responses towards MDM between serum-containing and serum-free media, but also correlated with the pro- and anti-tumoral nature of M1 and M2 type macrophages, respectively. Moreover, serum-free condition culture allowed the identification of tumor cell lines responsive to M2 conditioned media such as HCC1143 or T47D.


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)

Effect of monocyte-derived macrophage supernatant on HCC1143 cell line proliferation.Evaluation was performed in the presence or the absence of neutralizing TNF-α antibodies, in (A) conditioned media from M1 or M2 MDM cultured in RPMI 10% FBS or XVivo 10, (B) conditioned media from activated M1 (act. M1), M2a and M2c MDM stimulated in XVivo 10. Identical y-axis scales were used for comparison sake. Data represent mean ± SEM of three independent experiments including each condition in triplicate. Statistical significance was determined using t-test pairwise comparison (** p<0.005).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042656-g004: Effect of monocyte-derived macrophage supernatant on HCC1143 cell line proliferation.Evaluation was performed in the presence or the absence of neutralizing TNF-α antibodies, in (A) conditioned media from M1 or M2 MDM cultured in RPMI 10% FBS or XVivo 10, (B) conditioned media from activated M1 (act. M1), M2a and M2c MDM stimulated in XVivo 10. Identical y-axis scales were used for comparison sake. Data represent mean ± SEM of three independent experiments including each condition in triplicate. Statistical significance was determined using t-test pairwise comparison (** p<0.005).
Mentions: Both M1 and M2 conditioned media from culture in RPMI 10% FBS induced tumor cell growth inhibition at a similar level. However, different results were obtained when using CM from XVivo 10 culture. Indeed, M1 CM slightly reduced cell proliferation whereas M2 CM increased HCC1143 proliferation by 25% (Figure 4A). Decrease in cell proliferation was TNF-α independent as shown by the absence of effect upon addition of a neutralizing TNF-α antibody to the culture. We then assessed the effect of CM from activated M1, M2a and M2c culture on the tumor line viability (Figure 4B). HCC1143 proliferation was not affected in the presence of M2a CM whereas M2c increased cell growth by 25%. In activated M1 CM, HCC1143 proliferation was reduced by 60%. Inhibition of proliferation could be partially rescued by a neutralizing TNF-α antibody. IFN-γ and TGF-β neutralizing antibodies had no effect on tumor cell inhibition (data not shown). The data presented here were also confirmed using T47D and other tumor cell lines (Figure S1 and data not shown). Our data not only revealed significant differences in tumor cell line responses towards MDM between serum-containing and serum-free media, but also correlated with the pro- and anti-tumoral nature of M1 and M2 type macrophages, respectively. Moreover, serum-free condition culture allowed the identification of tumor cell lines responsive to M2 conditioned media such as HCC1143 or T47D.

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