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. Author manuscript; available in PMC: 2024 Aug 8.
Published in final edited form as: Oral Oncol. 2023 Sep 2;146:106562. doi: 10.1016/j.oraloncology.2023.106562

Figure 4. FGF/FGFR Family Genes are Highly Expressed and FGF Pathway Stimulation Enhances PD-L1 Expression in HNSCC.

Figure 4.

A, Expression heat map (Log2[FPKM+1]) for FGF/FGFR family genes in 40 HNSCC cell lines. B, Box and whisker plot (Log2[RSEM]) for expression of FGF/FGFR family genes in HNSCC TCGA dataset. C, UM-SCC-14a and UM-SCC-92 cells were treated +/− FGF2 (30 ng/mL), +/− IFNγ (10 ng/mL), or vehicle control (DMSO) for 72 hours. PD-L1 expression was assessed by western blot. D, UM-SCC-14a cells were seeded in a 6-well dish with a permeable membrane insert (0.4 μm pore size) upon which either additional UM-SCC-14a cells or UM-124-Fibroblasts were seeded as indicated. Cells were cultured for 72 hours followed by protein harvest and western blot for PD-L1 expression. E, UM-SCC-14a cells were seeded in a 6-well dish. Eighteen hours after seeding, media was left unchanged (untreated) or decanted and replaced with media collected from separate UM-SCC-14a cells or UM-124-Fibroblasts. Protein was harvested 24 hours after media change and PD-L1 expression was assessed by western blot. F, UM-SCC-14a cells were pre-treated with BGJ398 (5 μM), PD173074 (5 μM), or vehicle control (DMSO) in 1 mL of media for six hours. An additional 1 mL media was then added, either UM-124-Fibro conditioned media as in (E) or standard media +/− FGF2 (30 ng/mL), +/− IFNγ (10 ng/mL), or vehicle control (DMSO). Cells were then cultured for an additional 24 hours, followed by protein harvest and western blot for PD-L1. G, UM-SCC-14a cells overexpressing empty vector control (Puro-empty), FGFR1 wild-type (WT), or FGFR3 WT were treated +/− IFNγ (10 ng/mL), FGF2 (30 ng/mL), or vehicle control (DMSO) as indicated for 24 hours, followed by protein and RNA harvest. PD-L1 expression was assessed by western blot (G) and qPCR (H). RNA expression was assessed by qPCR in untreated samples only. I, UM-SCC-14a cells overexpressing empty vector control (GFP-empty), GFP-FGFR3-S249C mutant, or GFP-FGFR3-D786N mutant were treated +/− IFNγ (10 ng/mL), FGF2 (30 ng/mL), or vehicle control (DMSO) as indicated for 24 hours, followed by protein and RNA harvest. PD-L1 expression was assessed by western blot (I) and qPCR (J). RNA expression was assessed by qPCR in untreated samples only.