Figure 3 from Tumor-Intrinsic Activity of Chromobox 2 Remodels the Tumor Microenvironment in High-grade Serous Carcinoma

Modulation of CBX2 leads to shift in macrophage recruitment. A, OVCAR4 CBX2 OE and knockdown confirmation by qPCR. Diagram of protocol with direct co-culture of spheroids with primary monocytes. B, Confocal microscopy with infiltration of monocytes (green) increased with knockdown of CBX2. C, Confocal microscopy with infiltration of monocytes increased with overexpression of CBX2. D, Plots demonstrating the percentage of spheres with monocytes in shCBX2 or overexpression constructs. E, Plots demonstrating the number of spheres with CellTracker-positive monocytes in shCBX2 and overexpression. F, OVCAR4 (control) and CBX2 KO cells co-cultured with fluorescence-tagged primary monocytes (green, white arrowheads). Blue = nuclei. Images (top) are maximum projections of confocal z-stack and form-filling rendering brown, cancer cells; pink, monocytes (black arrowheads). 50-micron grid. G, Quantification of F. H, qPCR of CXCL1 and CXCL8 in siControl (siCTRL) and siCXCL1/siCXCL8. Internal control, HPRT. I, Flow cytometric analysis of digested co-culture spheroids to measure monocyte infiltration. J, Same as F, but with siControl and siCXCL1/8 in cells overexpressing CBX2. K, Quantification of J. Error bars, SEM. Statistical test, unpaired t test and multicomparison ANOVA.