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. 2022 Dec 13;11(2):199–216. doi: 10.1158/2326-6066.CIR-22-0410

Figure 2.

Figure 2. The correlation of circATP2B4 and M2 macrophages affects ovarian cancer progression. A, Expression of circATP2B4 was examined by ISH in normal ovarian epithelial tissues and EOC tissues. Scale bars, 50 μmol/L. B, ISH and multiplex fluorescent IHC of EOC nonmetastatic and metastatic tissue sections, DAB-circATP2B4, blue-DAPI, red-CD68, green-CD163, pink-CD206, representative images and IF with quantification. Scale bars, 20 μmol/L. C, TSNE plot showed the clusters of scRNA-seq in the ovarian cancer peritoneal metastasis samples from PRJNA609094 (GEO:GSE146026). Each dot is a cell colored by its analyzed cell types. D, Schematic illustration of the in vitro indirect coculture system. E, Macrophages (THP-1 with PMA) were cocultured with A2780-siNC or A2780-sicircATP2B4, or treated with control (PBS and IL4). After 48 hours, qRT-PCR was applied using primers for M2 markers (ARG1, IL10, CD163, CD206). The group treated with IL4 was used as positive control. F, Migration and invasion capacity of A2780 cocultured with macrophages (M, THP-1 with PMA) cocultured with A2780-siNC or A2780-sicircATP2B4, or treated with control (PBS and IL4) by the in vitro transwell coculture system. Scale bars, 300 μmol/L. G, Migration and invasion capacity of SKOV3 cocultured with macrophages (M, THP-1 with PMA) cocultured with SKOV3-pLCDH or SKOV3-circATP2B4, or treated with control (PBS and IL4) by the in vitro transwell coculture system. Representative photographs of migratory or invaded cells on the membrane coated with or without Matrigel (magnification, ×100) are shown. Scale bars, 300 μmol/L. H, The effect of the supernatants of macrophages cocultured with A2780-siNC or A2780-sicircATP2B4, or treated with control (PBS and IL4) on the EMT of A2780 was analyzed by Western blot analysis (*, P < 0.05). I, The effect of the supernatants of macrophages cocultured with SKOV3-pLCDH or SKOV3-circATP2B4, or treated with control (PBS and IL4) on the EMT of SKOV3 was analyzed by Western blot analysis. Densitometry shows relative protein expression normalized for β-actin. Data are representative of three independent experiments (*, P < 0.05). J, Kaplan–Meier survival curves in human ovarian cancer according to M2 macrophages infiltration abundance in TCGA-OV, AOCS, and GSE30161 data sets, (*, log-rank P < 0.05).

The correlation of circATP2B4 and M2 macrophages affects ovarian cancer progression. A, Expression of circATP2B4 was examined by ISH in normal ovarian epithelial tissues and EOC tissues. Scale bars, 50 μm. B, ISH and multiplex fluorescent IHC of EOC nonmetastatic and metastatic tissue sections, DAB-circATP2B4, blue-DAPI, red-CD68, green-CD163, pink-CD206, representative images and IF with quantification. Scale bars, 20 μm. C, TSNE plot showed the clusters of scRNA-seq in the ovarian cancer peritoneal metastasis samples from PRJNA609094 (GEO:GSE146026). Each dot is a cell colored by its analyzed cell types. D, Schematic illustration of the in vitro indirect coculture system. E, Macrophages (THP-1 with PMA) were cocultured with A2780-siNC or A2780-sicircATP2B4, or treated with control (PBS and IL4). After 48 hours, qRT-PCR was applied using primers for M2 markers (ARG1, IL10, CD163, CD206). The group treated with IL4 was used as positive control. F, Migration and invasion capacity of A2780 cocultured with macrophages (M, THP-1 with PMA) cocultured with A2780-siNC or A2780-sicircATP2B4, or treated with control (PBS and IL4) by the in vitro transwell coculture system. Scale bars, 300 μm. G, Migration and invasion capacity of SKOV3 cocultured with macrophages (M, THP-1 with PMA) cocultured with SKOV3-pLCDH or SKOV3-circATP2B4, or treated with control (PBS and IL4) by the in vitro transwell coculture system. Representative photographs of migratory or invaded cells on the membrane coated with or without Matrigel (magnification, ×100) are shown. Scale bars, 300 μm. H, The effect of the supernatants of macrophages cocultured with A2780-siNC or A2780-sicircATP2B4, or treated with control (PBS and IL4) on the epithelial-mesenchymal transition (EMT) of A2780 was analyzed by Western blot analysis (*, P < 0.05). I, The effect of the supernatants of macrophages cocultured with SKOV3-pLCDH or SKOV3-circATP2B4, or treated with control (PBS and IL4) on the EMT of SKOV3 was analyzed by Western blot analysis. Densitometry shows relative protein expression normalized for β-actin. Data are representative of three independent experiments (*, P < 0.05). J, Kaplan–Meier survival curves in human ovarian cancer according to M2 macrophages infiltration abundance in TCGA-OV, AOCS, and GSE30161 data sets. *, log-rank P < 0.05.