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Fig. 3 | Journal for ImmunoTherapy of Cancer

Fig. 3

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. 3

M2-polarized macrophages increase ALDH+ in CSC/M2 hetero-spheroids. a Elevated ALDH activity was assessed using the ALDEFLUOR flow cytometric analysis assay, with representative flow plots demonstrated. CSC/M2 hetero-spheroids, whether U937 or PBMC, have significantly (**p < 0.001, one-way ANOVA) elevated ALDH+ expression (1.8–2.4 fold). b Secreted pro-tumoral cytokine IL6 is elevated in hetero-spheroids compared to CSC mono-spheroids. c We suppressed signaling through the IL6/STAT3 axis using two inhibitors Ruxolitinib and sc144 in hetero-spheroids, and observed that this suppression also significantly (**p < 0.001, one-way ANOVA) decreased the ALDH+ enrichment within CSC/M2 hetero-spheroids. d Concomitant with the ALDH expression within hetero-spheroids, sensitivity to carboplatin was evaluated using the MTS assay. Normalized absorbance values indicated that CSC/M2 hetero-spheroids were significantly (*p < 0.05, one-way ANOVA) more resistant to carboplatin treatment compared to CSC mono-spheroids made with the same number of CSCs. e Representative phase contrast images indicate the loss of the tight spheroid boundaries in the CSC mono-spheroids in response to carboplatin treatment, indicative of cell death. The extent of the loss of boundary integrity is much lower in the CSC/M2 hetero-spheroids. Scale bar = 100 μm. f Quantification of the number of migrated cells in the lower chamber of a transwell system indicated that CSC/M2 hetero-spheroids were significantly (*p < 0.05, one-way ANOVA) more migratory compared to CSC mono-spheroids

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