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Fig. 4 | Journal for ImmunoTherapy of Cancer

Fig. 4

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. 4

Inhibition of macrophage Wnt-secretion reduces ALDH enrichment in hetero-spheroids. a Representative phase contrast image of a hetero-spheroid generated from CSCs and IWP-2 treated U937 M2 macrophages, indicates the formation of aggregated spheroids. Scale bar = 200 μm. b Flow analysis revealed that hetero-spheroids with IWP-2 treated M2 macrophages had a significantly diminished ALDH+ enrichment (**p < 0.001, one-way ANOVA) compared to control untreated CSC/M2 hetero-spheroids. However, no change in CD206 expression was observed in hetero-spheroids. c Transduction efficiency of monocytes with shRNA directed against WNT5B indicated a > 75% efficiency in knockdown of WNT5B gene expression in the shWnt5b targeted construct, and minimal changes in WNT5B gene expression in the scramble non-targeted lentiviral construct. d We utilized shWnt5b monocytes to differentiate and polarize M2 macrophages, and found that there was a 52% reduction in WNT5B gene expression in shWnt5b M2 macrophages, compared to scramble or control untreated M2 macrophages, indicating the knockdown of the Wnt5b gene. e No changes were observed in CD68 expression in monocytes generated from scramble or shWNT5B treated monocytes, demonstrated by flow analysis and representative plots. f Similarly, qPCR analysis of M2 gene expression markers CD163 and CD206 indicated increases in both genes, indicative of activation into the M2 program even in shWnt5b macrophages

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