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Fig. 5 | Journal for ImmunoTherapy of Cancer

Fig. 5

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. 5

Macrophage knockdown of Wnt5b suppresses ALDH enrichment, with no change in CD206, and increases chemosensitivity of hetero-spheroids. a Flow analysis of CD206 indicated no significant differences in CSC/M2 and CSC/Scramble M2 and CSC/shWnt5b M2 hetero-spheroids, indicating that CSCs were still capable of maintaining a robust immunosuppressive program in hetero-spheroids, regardless of WNT5B. b ALDH elevation however was significantly (*p < 0.05, one-way ANOVA) lowered compared to CSC/U937 M2 hetero-spheroids, and not significantly different (ns) compared to CSC mono-spheroids, indicating that knocking down Wnt5b expression in macrophages reduced ALDH enrichment in hetero-spheroids. c Concomitant with the decrease in ALDH, sensitivity to carboplatin also significantly (**p < 0.001, one-way ANOVA) improved, responding to treatment similar to CSC mono-spheroids. The red dashed line indicates the sensitivity levels of CSC mono-spheroids. d CSC/shWnt5b M2 hetero-spheroids were also significantly (***p < 0.0001, t-test) less invasive than CSC/Scramble M2 hetero-spheroids. e Upon ELISA analysis of secreted IL10 and IL6, we found no changes in IL-10 levels, while we found a stark decrease in secreted pro-tumoral IL-6 cytokine in CSC/shWnt5b M2 hetero-spheroids. f Exogenous addition of IL-6 (25 ng/ml) to CSC/shWnt5b M2 hetero-spheroids partially significantly (*p < 0.05, one-way ANOVA) increases ALDH+ enrichment, but does not restore it to levels of original enrichment with CSC/Scramble M2 or CSC/Ctrl M2 hetero-spheroids. g In order to identify if there was paracrine macrophage Wnt-driven Wnt signaling in CSCs, we separated the CSCs (using a GFP label) from hetero-spheroids, and probed for gene expression of several Wnt ligands we saw elevated in CSCs compared to bulk OVCAR3. We observed that with M2 macrophage co-culture, several Wnt ligands were upregulated (Wnt2 significantly elevated, ***p < 0.001, two-way ANOVA). However, this elevated gene expression of Wnt ligands was lost in CSCs in hetero-spheroid culture with shWnt5b M2, indicating that Wnt5b was partly responsible for potentiating Wnt signaling within the CSC compartment. h This loss in Wnt ligand expression also translated to decreased β-catenin expression in CSC/shWnt5b M2 hetero-spheroids compared to CSC/U937 M2 hetero-spheroids, quantified by densitometry of immunoblots (representative blot shown)

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