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. 2024 Jan 8;8:7. doi: 10.1038/s41698-023-00495-5

Fig. 2. CAFs regulate OCSCs.

Fig. 2

A, B ALDEFLUOR assay for stem cell enrichment in OC-CAF coculture. OVCAR3/Kuramochi cells were seeded with CAFs and cocultured for a week. ALDEFLUOR assay was performed to label CSC (green). A Fluorescent imaging of OC-CAF coculture labeled by ALDEFLUOR. Scale bar: 100 μm. B Flow cytometry analysis was done to quantify CSCs in control/CAF cocultured group. Mean ± SD from three independent experiments. *p < 0.01 (t-test). C Spheroid formation assay of OC-CAF coculture. OVCAR3/Kuramochi cells were seeded with/without CAFs in ultra-low adhesion plates and cocultured for 14 days. Images and quantification of number of spheroids are shown as fold change compared to the respective OC cell alone. Scale bar: 400 μm. Mean ± SD from three independent experiments. *p < 0.01 (t-test). D, E Limiting dilution assay for tumor initiation frequency. OVCAR3 cells were cocultured with RFP expressing CAFs for 7 days, followed by isolation using FACS, then subcutaneously injected into the right flank of female NSG mice. Control, sham treated OVCAR3 cells were injected into the left flank of the same mouse. D Schematic of the experiment plan (left) and the tumor formation and tumor-initiating cell frequency calculated by extreme limiting dilution analysis (ELDA) are shown (right). E Representative IHC Images of the xenograft tumor sections stained with ALDH1A1. Scale bar: 400 μm. F ALDEFLUOR assay for stem cell enrichment in OC-CAF coculture using OC patient derived ascites cells. Ascites derived ovarian cancer (AOC) cells were seeded with/without CAFs and cocultured for a week. ALDEFLUOR assay was performed to label CSC (green). Flow cytometry analysis was done to quantify CSCs in control/CAF cocultured groups. Mean ± SD from three independent experiments. **p < 0.05 (t-test). G Spheroid formation assay using AOCs. AOCs were seeded with/without CAFs in ultra-low adhesion plates and cocultured for 14 days. Images and quantifications are shown. Scale bar: 400 μm. Mean ± SD from three independent experiments. *p < 0.01 (t-test). H–K ALDEFLUOR assay of OC-CAF coculture for stem cell enrichment using pure CSC/non-CSC. Following ALDEFLUOR assay, the OVCAR3 cells were sorted by FACS to isolate pure ALDH+ and ALDH- cells. The ALDH+ and ALDH- cells were then seeded with CAFs and cocultured for a week. ALDEFLUOR assay was again performed to label CSC (green). Fluorescent imaging (H, J) and flow cytometric analysis (I, K) of ALDH+ (H–I) and ALDH- (J, K) were shown. Scale bar: 100 μm. Mean ± SD from three independent experiments. *p < 0.01 (t-test).