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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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Phagocytic activity of M1, M2a and M2c monocyte-derived macrophages towards MOLT4.Gating strategy used to assess phagocytosis of MOLT4 debris by MDM obtained by flow cytometry: controls included assessment of MOLT4 alone or together with MDM in PBS (A). Dot plots representing data from two distinct donors in XVivo 10 as described in Methods (B). Mean fluorescence intensity (median) of phagocytosing MDM derived from ten distinct donors, attributed with identical (□) or differential (• or ○) activity between activated M1 and M2a or M2c MDM (C). Horizontal bars represent the median value of each group.
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pone-0042656-g005: Phagocytic activity of M1, M2a and M2c monocyte-derived macrophages towards MOLT4.Gating strategy used to assess phagocytosis of MOLT4 debris by MDM obtained by flow cytometry: controls included assessment of MOLT4 alone or together with MDM in PBS (A). Dot plots representing data from two distinct donors in XVivo 10 as described in Methods (B). Mean fluorescence intensity (median) of phagocytosing MDM derived from ten distinct donors, attributed with identical (□) or differential (• or ○) activity between activated M1 and M2a or M2c MDM (C). Horizontal bars represent the median value of each group.

Mentions: Since only the M1 and M2 MDM differentiated and activated in serum-free conditions reflected both MDM pro and anti-tumoral activity, we studied their phagocytic activity towards leukaemia cell line MOLT4 debris. Leukaemia cell lines have been used before to study antibody-independent MDM phagocytosis [24]. Target cells were stained with green membrane dye CMFDA, killed by repeated freeze/thaw cycles and incubated for 4 h together with MDM in XVivo 10 or PBS serving as a negative control. Uptake of target cells (DAPI+ CD11b−) by MDM was monitored by flow cytometry and gating was performed on live MDM (DAPI− CD11b+), as represented in Figure 5A. The frequency of phagocytosed MOLT4 particles was defined by the proportion of CD11b+ MDM that acquired green fluorescence (Figure 5B, upper quadrant). Activated M1, M2a and M2c MDM from ten different donors were tested and the proportion of CD11b+ Green CMFDA+ effector cells represented 5 to 80% MDM regardless of the stimulation, indicating high variability in phagocytosis activity among donors. When analyzing the median fluorescence intensity of phagocytosing cells, MDM could be divided into three groups with regards to differences in activity between M1and M2a or M2c (Figure 5C). In the first one, M2a and M2c MDM derived from six donors, among which donor 1 (depicted in Figure 5B), exhibited greater phagocytic properties compared to activated M1 MDM. The second group consisted of one donor which activated M1 MDM showed higher phagocytic activity than M2a or M2c MDM whereas the third group included activated M1 and M2 MDM, derived from three donors, displaying identical functionality such as in donor 2. In summary, our data demonstrated that M2a and M2c MDM exhibited greater phagocytic activity compared to activated M1 MDM despite individual donor variability, correlating with the scavenging properties of M2 macrophages expressing higher level of surface receptors involved in debris clearance compared to M1 MDM.


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)

Phagocytic activity of M1, M2a and M2c monocyte-derived macrophages towards MOLT4.Gating strategy used to assess phagocytosis of MOLT4 debris by MDM obtained by flow cytometry: controls included assessment of MOLT4 alone or together with MDM in PBS (A). Dot plots representing data from two distinct donors in XVivo 10 as described in Methods (B). Mean fluorescence intensity (median) of phagocytosing MDM derived from ten distinct donors, attributed with identical (□) or differential (• or ○) activity between activated M1 and M2a or M2c MDM (C). Horizontal bars represent the median value of each group.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042656-g005: Phagocytic activity of M1, M2a and M2c monocyte-derived macrophages towards MOLT4.Gating strategy used to assess phagocytosis of MOLT4 debris by MDM obtained by flow cytometry: controls included assessment of MOLT4 alone or together with MDM in PBS (A). Dot plots representing data from two distinct donors in XVivo 10 as described in Methods (B). Mean fluorescence intensity (median) of phagocytosing MDM derived from ten distinct donors, attributed with identical (□) or differential (• or ○) activity between activated M1 and M2a or M2c MDM (C). Horizontal bars represent the median value of each group.
Mentions: Since only the M1 and M2 MDM differentiated and activated in serum-free conditions reflected both MDM pro and anti-tumoral activity, we studied their phagocytic activity towards leukaemia cell line MOLT4 debris. Leukaemia cell lines have been used before to study antibody-independent MDM phagocytosis [24]. Target cells were stained with green membrane dye CMFDA, killed by repeated freeze/thaw cycles and incubated for 4 h together with MDM in XVivo 10 or PBS serving as a negative control. Uptake of target cells (DAPI+ CD11b−) by MDM was monitored by flow cytometry and gating was performed on live MDM (DAPI− CD11b+), as represented in Figure 5A. The frequency of phagocytosed MOLT4 particles was defined by the proportion of CD11b+ MDM that acquired green fluorescence (Figure 5B, upper quadrant). Activated M1, M2a and M2c MDM from ten different donors were tested and the proportion of CD11b+ Green CMFDA+ effector cells represented 5 to 80% MDM regardless of the stimulation, indicating high variability in phagocytosis activity among donors. When analyzing the median fluorescence intensity of phagocytosing cells, MDM could be divided into three groups with regards to differences in activity between M1and M2a or M2c (Figure 5C). In the first one, M2a and M2c MDM derived from six donors, among which donor 1 (depicted in Figure 5B), exhibited greater phagocytic properties compared to activated M1 MDM. The second group consisted of one donor which activated M1 MDM showed higher phagocytic activity than M2a or M2c MDM whereas the third group included activated M1 and M2 MDM, derived from three donors, displaying identical functionality such as in donor 2. In summary, our data demonstrated that M2a and M2c MDM exhibited greater phagocytic activity compared to activated M1 MDM despite individual donor variability, correlating with the scavenging properties of M2 macrophages expressing higher level of surface receptors involved in debris clearance compared to M1 MDM.

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