Journal: Cells

Article Title: Regulatory T Cells Induce Metastasis by Activating Tgf-Β and Enhancing the Epithelial–Mesenchymal Transition

doi: 10.3390/cells8111387

Figure Lengend Snippet: Increased migration of cancer cells and Tregs in both tumors established from Treg-co-cultured B16-BL6 cells or tumors injected with Tregs. To assess the effect of Tregs on the migration of dissociated tumor cells, ( A ) B16-BL6 cells were co-cultured with Tregs for 72 h at co-culture ratios of 1:10 when inadherent Tregs were removed from culture by multiple washing steps. Subsequently, the cells were detached and 5 × 10 5 cells were injected subcutaneously into the right abdomen to establish a tumor or ( B ) subcutaneous tumors in the right abdomen were established using naïve B16-BL6 cells and subsequently injected intratumorally with Tregs (2 × 10 7 cells) three times every other day. The migration profile of cells dissociated from the B16-BL6 tumors was analyzed using a modified transwell migration chamber. The cells that had migrated to the lower filter surface were stained with H & E. The images are representatives of results from 3 independent experiments. Original magnification: ×200. Migrated cells were counted in three randomly selected fields. Data are shown as mean ± SD of results from three independent fields/well. *** p < 0.001 or ** p < 0.01. ( C ) Tumors were collected from mice injected with Tregs at 5 days after the final injection. The populations of Tregs in tumors from mice were analyzed by flow cytometry. Gating was for cluster of differentiation (CD)4 + T cells and analysis for CD25 + and Foxp3 + cells. The data are representative of three independent experiments performed in triplicate. Data points are mean ± SD ( n = 3 per group). *** p < 0.001.

Article Snippet: Samples were analysed using a BD Biosciences BD FACScanto II flow cytometry analyser and FACSDiva software (BD Biosciences).

Techniques: Migration, Cell Culture, Injection, Co-Culture Assay, Modification, Staining, Flow Cytometry