FIGURE 2.

Cancer‐associated fibroblasts inhibit immune cell infiltrate. (A) A375 cell cluster and A375‐fibroblast bicellular spheroids. Melanoma cells and fibroblasts were mixed at a 1:2 ratio and seeded on agarose. Representative images of A375 cell cluster and A375‐fibroblast spheroids at days 1, 3, 5 and 7 were shown. Scale bar, 250 μm. (B) Representative fluorescent images of A375 cell cluster and A375‐fibroblast spheroid. Tumour cells in the spheroids were labelled with CellTracker™ Orange CMRA dye (red), and the fibroblast cells were labelled with CFSE (green). Scale bar, 200 μm. (C) PD‐L1 expression in response to IFN‐γ on A375 cells, fibroblasts and CAFs in A375‐fibroblast spheroids. A375, fibroblasts and A375‐fibroblast spheroids were cultured with 50 ng/ml IFN‐γ for 24 h and PD‐L1 expression on A375, fibroblasts and CAFs was measured by FACS. (D) Representative images of A375 cell cluster and A375‐fibroblast spheroids co‐cultured with peripheral blood mononuclear cells (PBMCs). Melanoma cells and fibroblasts were labelled with orange CMRA dye (red), and PBMCs were labelled with CFSE (green). Scale bar, 200 μm. (E) Infiltrating γδ T cells in the A375 and A375‐fibroblast spheroids were measured by FACS. Spheroids were co‐cultured with PBMCs for 24 h and CFSE labelled immune cells were quantified by FACS. n = 5; **p < 0.01. (F) Average γδ T cells, CD4⁺ T cells, CD8⁺ T cells and CD11b⁺ cells outside or inside of an A2058 tumour spheroid (T‐out or T‐in) and A2058‐fibroblast spheroid (F‐out or F‐in) were measured by FACS. * p < 0.05. (G) The CTLA‐4, PD‐1, PD‐L1 and CD69 expression on γδ T cells outside or inside of A2058 tumour spheroid (T‐out or T‐in) and A2058‐fibroblast spheroids (F‐out or F‐in). * p < 0.05. **p < 0.01. n = 3–7. Con: PBMCs cultured without tumour spheroids, T‐out: outside of unicellular tumour spheroids, T‐in: inside of unicellular tumour spheroids, F‐out: outside of A2058‐fibroblast spheroids, F‐in: inside of A2058‐fibroblast spheroids