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Macrophages inhibit human osteosarcoma cell growth after activation with the bacterial cell wall derivative liposomal muramyl tripeptide in combination with interferon-γ.

Pahl JH, Kwappenberg KM, Varypataki EM, Santos SJ, Kuijjer ML, Mohamed S, Wijnen JT, van Tol MJ, Cleton-Jansen AM, Egeler RM, Jiskoot W, Lankester AC, Schilham MW - J. Exp. Clin. Cancer Res. (2014)

Bottom Line: Antibody-dependent effects were investigated using the therapeutic anti-EGFR antibody cetuximab.Addition of the tumor-reactive anti-EGFR antibody cetuximab did not further improve the anti-tumor activity of activated M1-like macrophages.However, specific blockage of these cytokines, nitric oxide or reactive oxygen species did not inhibit the anti-tumor effect, suggesting the involvement of other soluble factors released upon macrophage activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Leiden University Medical Center, Leiden, the Netherlands. M.W.Schilham@lumc.nl.

ABSTRACT

Background: In osteosarcoma, the presence of tumor-infiltrating macrophages positively correlates with patient survival in contrast to the negative effect of tumor-associated macrophages in patients with other tumors. Liposome-encapsulated muramyl tripeptide (L-MTP-PE) has been introduced in the treatment of osteosarcoma patients, which may enhance the potential anti-tumor activity of macrophages. Direct anti-tumor activity of human macrophages against human osteosarcoma cells has not been described so far. Hence, we assessed osteosarcoma cell growth after co-culture with human macrophages.

Methods: Monocyte-derived M1-like and M2-like macrophages were polarized with LPS + IFN-γ, L-MTP-PE +/- IFN-γ or IL-10 and incubated with osteosarcoma cells. Two days later, viable tumor cell numbers were analyzed. Antibody-dependent effects were investigated using the therapeutic anti-EGFR antibody cetuximab.

Results: M1-like macrophages inhibited osteosarcoma cell growth when activated with LPS + IFN-γ. Likewise, stimulation of M1-like macrophages with liposomal muramyl tripeptide (L-MTP-PE) inhibited tumor growth, but only when combined with IFN-γ. Addition of the tumor-reactive anti-EGFR antibody cetuximab did not further improve the anti-tumor activity of activated M1-like macrophages. The inhibition was mediated by supernatants of activated M1-like macrophages, containing TNF-α and IL-1β. However, specific blockage of these cytokines, nitric oxide or reactive oxygen species did not inhibit the anti-tumor effect, suggesting the involvement of other soluble factors released upon macrophage activation. While LPS + IFN-γ-activated M2-like macrophages had low anti-tumor activity, IL-10-polarized M2-like macrophages were able to reduce osteosarcoma cell growth in the presence of the anti-EGFR cetuximab involving antibody-dependent tumor cell phagocytosis.

Conclusion: This study demonstrates that human macrophages can be induced to exert direct anti-tumor activity against osteosarcoma cells. Our observation that the induction of macrophage anti-tumor activity by L-MTP-PE required IFN-γ may be of relevance for the optimization of L-MTP-PE therapy in osteosarcoma patients.

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Inhibition of osteosarcoma cell growth by LPS + IFN-γ–activated M1-like macrophages. Human M1-like macrophages (dark shade) and M2-like macrophages (light shade) were pre-activated with or without LPS + IFN-γ (M1 and M2) or IL-10 (M2) and afterwards incubated with (A) HOS-143b cells (n = 4–11) and (B) OHS cells (n = 5–12) and (C) four other osteosarcoma cell lines (n = 2–12). After two days of co-culture, tumor cell numbers were counted by flow cytometry. Differences between one macrophage–tumor co-culture and the control, i.e. tumor cell recovery after incubation in the absence of macrophages (white bar, set to 100%), as in panel C were statistically analyzed by paired student t-tests, *** is P < 0.001, ** is P < 0.01, * is P < 0.05, ns is not statistically significant. Differences between multiple groups as in panel A and B were statistically analyzed by ANOVA as indicated followed by Dunnett’s post test for differences (p < 0.05) between individual co-cultures and the control as indicated by asterisks. (D) HOS-143b cell counts after 2, 24 and 48 hours co-culture with M1-like macrophages +/− LPS + IFN-γ (n = 5). All data are means of multiple experiments as indicated (n).
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Figure 1: Inhibition of osteosarcoma cell growth by LPS + IFN-γ–activated M1-like macrophages. Human M1-like macrophages (dark shade) and M2-like macrophages (light shade) were pre-activated with or without LPS + IFN-γ (M1 and M2) or IL-10 (M2) and afterwards incubated with (A) HOS-143b cells (n = 4–11) and (B) OHS cells (n = 5–12) and (C) four other osteosarcoma cell lines (n = 2–12). After two days of co-culture, tumor cell numbers were counted by flow cytometry. Differences between one macrophage–tumor co-culture and the control, i.e. tumor cell recovery after incubation in the absence of macrophages (white bar, set to 100%), as in panel C were statistically analyzed by paired student t-tests, *** is P < 0.001, ** is P < 0.01, * is P < 0.05, ns is not statistically significant. Differences between multiple groups as in panel A and B were statistically analyzed by ANOVA as indicated followed by Dunnett’s post test for differences (p < 0.05) between individual co-cultures and the control as indicated by asterisks. (D) HOS-143b cell counts after 2, 24 and 48 hours co-culture with M1-like macrophages +/− LPS + IFN-γ (n = 5). All data are means of multiple experiments as indicated (n).

Mentions: The potential of human macrophages to inhibit osteosarcoma cell growth in vitro was investigated. M1-like and M2-like macrophages were differentiated from blood monocytes with or without the polarization stimuli LPS + IFN-γ (for M1 and M2) or IL-10 (for M2) as previously established[10,26,28]. The various macrophage subtypes were co-cultured with osteosarcoma cell lines and after two days the residual number of viable tumor cells was assessed by flow cytometry[15,27]. In particular M1-like macrophages pre-stimulated with LPS + IFN-γ were able to significantly reduce tumor cell numbers of HOS-143b and OHS cells to as low as 50% and to lesser extend of four other osteosarcoma cell lines (Figure 1, panel A-C). The inhibition of tumor cell growth as a consequence of macrophage addition was not yet apparent after 2 and 24 hours of co-culture but became pronounced after two days of co-culture (Figure 1, panel D). The inhibitory effect of activated M1-like macrophages could be titrated and was near-maximal at >6:1 ratio (Additional file1: Figure S1, panel A).


Macrophages inhibit human osteosarcoma cell growth after activation with the bacterial cell wall derivative liposomal muramyl tripeptide in combination with interferon-γ.

Pahl JH, Kwappenberg KM, Varypataki EM, Santos SJ, Kuijjer ML, Mohamed S, Wijnen JT, van Tol MJ, Cleton-Jansen AM, Egeler RM, Jiskoot W, Lankester AC, Schilham MW - J. Exp. Clin. Cancer Res. (2014)

Inhibition of osteosarcoma cell growth by LPS + IFN-γ–activated M1-like macrophages. Human M1-like macrophages (dark shade) and M2-like macrophages (light shade) were pre-activated with or without LPS + IFN-γ (M1 and M2) or IL-10 (M2) and afterwards incubated with (A) HOS-143b cells (n = 4–11) and (B) OHS cells (n = 5–12) and (C) four other osteosarcoma cell lines (n = 2–12). After two days of co-culture, tumor cell numbers were counted by flow cytometry. Differences between one macrophage–tumor co-culture and the control, i.e. tumor cell recovery after incubation in the absence of macrophages (white bar, set to 100%), as in panel C were statistically analyzed by paired student t-tests, *** is P < 0.001, ** is P < 0.01, * is P < 0.05, ns is not statistically significant. Differences between multiple groups as in panel A and B were statistically analyzed by ANOVA as indicated followed by Dunnett’s post test for differences (p < 0.05) between individual co-cultures and the control as indicated by asterisks. (D) HOS-143b cell counts after 2, 24 and 48 hours co-culture with M1-like macrophages +/− LPS + IFN-γ (n = 5). All data are means of multiple experiments as indicated (n).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4007518&req=5

Figure 1: Inhibition of osteosarcoma cell growth by LPS + IFN-γ–activated M1-like macrophages. Human M1-like macrophages (dark shade) and M2-like macrophages (light shade) were pre-activated with or without LPS + IFN-γ (M1 and M2) or IL-10 (M2) and afterwards incubated with (A) HOS-143b cells (n = 4–11) and (B) OHS cells (n = 5–12) and (C) four other osteosarcoma cell lines (n = 2–12). After two days of co-culture, tumor cell numbers were counted by flow cytometry. Differences between one macrophage–tumor co-culture and the control, i.e. tumor cell recovery after incubation in the absence of macrophages (white bar, set to 100%), as in panel C were statistically analyzed by paired student t-tests, *** is P < 0.001, ** is P < 0.01, * is P < 0.05, ns is not statistically significant. Differences between multiple groups as in panel A and B were statistically analyzed by ANOVA as indicated followed by Dunnett’s post test for differences (p < 0.05) between individual co-cultures and the control as indicated by asterisks. (D) HOS-143b cell counts after 2, 24 and 48 hours co-culture with M1-like macrophages +/− LPS + IFN-γ (n = 5). All data are means of multiple experiments as indicated (n).
Mentions: The potential of human macrophages to inhibit osteosarcoma cell growth in vitro was investigated. M1-like and M2-like macrophages were differentiated from blood monocytes with or without the polarization stimuli LPS + IFN-γ (for M1 and M2) or IL-10 (for M2) as previously established[10,26,28]. The various macrophage subtypes were co-cultured with osteosarcoma cell lines and after two days the residual number of viable tumor cells was assessed by flow cytometry[15,27]. In particular M1-like macrophages pre-stimulated with LPS + IFN-γ were able to significantly reduce tumor cell numbers of HOS-143b and OHS cells to as low as 50% and to lesser extend of four other osteosarcoma cell lines (Figure 1, panel A-C). The inhibition of tumor cell growth as a consequence of macrophage addition was not yet apparent after 2 and 24 hours of co-culture but became pronounced after two days of co-culture (Figure 1, panel D). The inhibitory effect of activated M1-like macrophages could be titrated and was near-maximal at >6:1 ratio (Additional file1: Figure S1, panel A).

Bottom Line: Antibody-dependent effects were investigated using the therapeutic anti-EGFR antibody cetuximab.Addition of the tumor-reactive anti-EGFR antibody cetuximab did not further improve the anti-tumor activity of activated M1-like macrophages.However, specific blockage of these cytokines, nitric oxide or reactive oxygen species did not inhibit the anti-tumor effect, suggesting the involvement of other soluble factors released upon macrophage activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Leiden University Medical Center, Leiden, the Netherlands. M.W.Schilham@lumc.nl.

ABSTRACT

Background: In osteosarcoma, the presence of tumor-infiltrating macrophages positively correlates with patient survival in contrast to the negative effect of tumor-associated macrophages in patients with other tumors. Liposome-encapsulated muramyl tripeptide (L-MTP-PE) has been introduced in the treatment of osteosarcoma patients, which may enhance the potential anti-tumor activity of macrophages. Direct anti-tumor activity of human macrophages against human osteosarcoma cells has not been described so far. Hence, we assessed osteosarcoma cell growth after co-culture with human macrophages.

Methods: Monocyte-derived M1-like and M2-like macrophages were polarized with LPS + IFN-γ, L-MTP-PE +/- IFN-γ or IL-10 and incubated with osteosarcoma cells. Two days later, viable tumor cell numbers were analyzed. Antibody-dependent effects were investigated using the therapeutic anti-EGFR antibody cetuximab.

Results: M1-like macrophages inhibited osteosarcoma cell growth when activated with LPS + IFN-γ. Likewise, stimulation of M1-like macrophages with liposomal muramyl tripeptide (L-MTP-PE) inhibited tumor growth, but only when combined with IFN-γ. Addition of the tumor-reactive anti-EGFR antibody cetuximab did not further improve the anti-tumor activity of activated M1-like macrophages. The inhibition was mediated by supernatants of activated M1-like macrophages, containing TNF-α and IL-1β. However, specific blockage of these cytokines, nitric oxide or reactive oxygen species did not inhibit the anti-tumor effect, suggesting the involvement of other soluble factors released upon macrophage activation. While LPS + IFN-γ-activated M2-like macrophages had low anti-tumor activity, IL-10-polarized M2-like macrophages were able to reduce osteosarcoma cell growth in the presence of the anti-EGFR cetuximab involving antibody-dependent tumor cell phagocytosis.

Conclusion: This study demonstrates that human macrophages can be induced to exert direct anti-tumor activity against osteosarcoma cells. Our observation that the induction of macrophage anti-tumor activity by L-MTP-PE required IFN-γ may be of relevance for the optimization of L-MTP-PE therapy in osteosarcoma patients.

Show MeSH
Related in: MedlinePlus