Tumor cells expressed EpCAM and could be detected in monoculture as well as co-culture with all the cell lines. found within the spheroid as well. FACS analysis of cell populations in co-culture spheroids was performed on day 5. Cells were cultured as indicated earlier. Spheroids were collected and treated with cell dissociation reagent to get single cells for the analysis. Cell suspensions were incubated with Procyanidin B2 anti-FAP antibody (activated fibroblast/ marker) or with anti-EpCAM antibody (Epithelial cell marker). Tumor cells expressed EpCAM and could be detected in monoculture as well as co-culture with all the cell lines. However, few or no fibroblasts could be detected on day 5 indicating that even though initially more fibroblasts Mouse monoclonal to CD16.COC16 reacts with human CD16, a 50-65 kDa Fcg receptor IIIa (FcgRIII), expressed on NK cells, monocytes/macrophages and granulocytes. It is a human NK cell associated antigen. CD16 is a low affinity receptor for IgG which functions in phagocytosis and ADCC, as well as in signal transduction and NK cell activation. The CD16 blocks the binding of soluble immune complexes to granulocytes were added than tumor cells, the co-culture conditions favored tumor cell proliferation.(TIF) pone.0127948.s002.tif (480K) GUID:?2C81F8F9-4618-4E2B-A94C-F18408F9298A S3 Fig: GC profiles of the MRC5 and LT2 fibroblasts and the primary TAFs. The supernatants from mono-cultured fibroblast spheroids were collected on day 5, and 42 different growth factors and cytokines were measured using Luminex multiplex technology. The growth factors and cytokines that were produced at detectable levels are depicted in the graph. Among these growth factors, the lung fibroblast cell lines MRC5 and 129A produced higher levels of HGF and VEGF than the pancreatic fibroblast cell line LT2 and the primary breast TAF cell line 161A. The LT2 cells secreted higher levels of PDGF than the other fibroblast cell types. Regarding the cytokines, all of the fibroblasts secreted high levels of IL6 and IL8. The expression of MCSF was higher in 161A breast TAFs than in the other fibroblast cell types. The LT2 pancreatic fibroblasts produced higher levels of G-CSF and GM-CSF than the other fibroblast cell types.(TIF) pone.0127948.s003.tif (258K) GUID:?708C0684-E3D7-4FA4-8DD5-D3C3CD58F77E S4 Fig: GC profiles of the tumor cell-MRC5 fibroblast co-cultures. The supernatants from co-culture spheroids were collected on day 5, and 42 different growth factors and cytokines were Procyanidin B2 measured using Luminex multiplex technology. The growth factors and cytokines that were produced at detectable levels Procyanidin B2 are depicted in the graph.(TIF) pone.0127948.s004.tif (154K) GUID:?A053A679-9D9D-4C0C-8230-CAC2FE3147DD S5 Fig: Differential expression and activation of EGFR, cMet and STAT3 in the 3D co-cultures. Cancer cells and fibroblasts (MRC5) were cultured as either monocultures or co-cultures for 5 days as described in the cell viability assay. On day 5, spheroids were collected, and lysates were prepared for Western blot. A. The expression of EGFR and phospho-EGFR, the activated Procyanidin B2 form of EGFR, was detected in the Bxpc3 lysates using specific antibodies. Although the EGFR levels were only slightly increased in the co-cultures with the MRC5 cells, the expression of the phosphorylated form of EGFR was clearly increased in the co-cultures compared to the monocultures. B. The expression of cMet was detectable In H596 cells that were monocultured as well as those that were co-cultured with MRC5 cells. However, the cMet expression level was higher in the co-cultures, and the expression of phospho-cMet was only detected in the co-cultures. C. Although the monocultured BT20 cells expressed STAT3, they did not exhibit the activation of this factor. The level of p-STAT3 was increased in the co-cultured BT20 cells and was also detectable in the monocultured fibroblasts.(TIF) pone.0127948.s005.tif (225K) GUID:?B9BA32AC-16B9-478A-88B2-1BEF77D7A6DD S6 Fig: Growth factor secretion by cell lines that were not dependent on fibroblast co-culture for survival. The supernatants from co-culture spheroids of cell lines that were not dependent on fibroblast co-culture for survival were collected on day 5, and 42 different growth factors and cytokines were measured using Luminex multiplex technology. The growth factors and cytokines that were produced at detectable levels are depicted in the graph.(TIF) pone.0127948.s006.tif (63K) GUID:?2B4A1DA4-F77B-4F90-826C-E67E8D43A6EC Abstract In recent years, evidence has indicated that the tumor microenvironment (TME) plays a significant role in tumor progression. Fibroblasts represent an abundant cell population in the TME and produce several growth factors and cytokines. Fibroblasts generate a suitable niche for tumor cell survival and metastasis under the influence of interactions between fibroblasts and tumor cells. Investigating these.