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Fig. 2 | Journal for ImmunoTherapy of Cancer

Fig. 2

From: Ovarian cancer stem cells and macrophages reciprocally interact through the WNT pathway to promote pro-tumoral and malignant phenotypes in 3D engineered microenvironments

Fig. 2

Hetero-spheroids drive CD206 polarization in monocyte-derived macrophages. a Hetero-spheroids were generated from bulk unsorted OVCAR3 cells and M0 macrophages (OVCAR3/U937 M0), and CSC/U937 M0. Flow analysis for the macrophage polarization marker CD206 indicated that while OVCAR3 and CSCs by themselves do not express CD206, CSCs drive CD206 expression in previously CD206 M0 macrophages within hetero-spheroids at significantly (**p < 0.001, one-way ANOVA) higher levels than OVCAR3. Representative flow analysis plots are shown. b IL10 gene expression was evaluated using qPCR in mono- and hetero-spheroids, indicating that CSCs had a 2-fold increased gene expression of IL10, likely driving CD206 expression in M0 macrophages to higher extents than OVCAR3. c CD206 expression was maintained at elevated levels (18.5–29.24%) in all CSC hetero-spheroid conditions, indicating that the activated M2 phenotype was maintained in macrophages within hetero-spheroids. d Levels of secreted IL-10 were mildly elevated in CSC/M2 hetero-spheroids, but were also similar across all CSC/macrophage hetero-spheroids, indicating the presence of the immuno-suppressive cytokine. e When CSCs were pre-treated with the Wnt secretion inhibitor, IWP-2, significantly lower (*p < 0.05,one-way ANOVA) CD206 expression was observed in CSC/M2 hetero-spheroids, implying the importance of CSC-derived Wnt ligands in the maintenance of M2 activation in macrophages. f qPCR analysis revealed that gene expression of several Wnt ligands were elevated in CSC compared to bulk OVCAR3 spheroids (dotted line = no change); (g) Densitometric measurements of immunoblots for β-catenin compared to the loading control β-actin indicated that CSC/M2 spheroids had a 26% increase in β-catenin expression, compared to CSC mono-spheroids, indicating higher Wnt/β-catenin activity in CSC/M2 hetero-spheroids

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