Fibroblast activation and monocyte TAM polarization in tumorigenic multicellular 3D spheroids. (A) Schematic diagram representing isolation of fibroblasts from 3D multicellular spheroids using anti-fibroblast microbeads-based cell sorting to examine CAF markers such as α-SMA, FSP/S100A4, and PDGF-β. (B–D) RT-qPCR analysis shows a relative abundance of α-SMA (B), FSP (C), and PDGF-β (D) mRNA levels in isolated fibroblasts of BMT, AMT, and HMT spheroids on day 0, day 7, and day 10. (E) Immunofluorescence staining of α-SMA in BMT, AMT, and HMT spheroid sections (6 micron). DAPI was used for nuclei counterstaining. (F) Schematic diagram representing isolation of macrophages from 3D multicellular spheroids using anti-human CD11b microbeads to examine TAM markers CD163 and CD206. (G) Flow cytometric analysis and quantification of CD163+ cells in BMT, AMT, and HMT spheroids on day 10. (H, I) RT-qPCR analysis of CD206 (H) and CD163 (I) gene expression in macrophages isolated from BMT, AMT, and HMT spheroids on day 0, day 7, and day 10. (J) Immunofluorescence staining of CD163 in BMT, AMT, and HMT spheroid sections (6 micron). DAPI was used for nuclei counterstaining. GAPDH is used as a loading control for RT-qPCR analysis. Fold change for CAF markers was calculated keeping MRC5 as control cells and fold change for TAM population was calculated keeping THP-1 cells as control cells. All experiments were performed in triplicate (n=3). Data represented as mean ± S.D. 2D vs BMT over time *p< 0.05, **p< 0.01, ***p< 0.001; BMT vs AMT over time ##p< 0.01, ###p< 0.001; BMT vs HMT over time §§p< 0.01, §§§p< 0.001; ns, non-significant.
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