Figure 2.

Verification of microarray with qRT-PCR. Five head and neck squamous cell carcinoma cell lines (LK0824, LK0858, LK0923, LK0942, LK0949) were grown alone or co-cultured with their patient-matched cancer-associated fibroblasts (CAFs) as 2D monolayers for 7 days. RNA was isolated from tumor cells (separated from CAFs by magnetic activated cell sorting) and the mRNA expression of the COL1A2, GREM1, and POSTN genes, which, by microarray analysis, were identified as differentially expressed in co-cultures, was analyzed by qRT-PCR. The log₂ mRNA levels of co-cultured tumor cells are shown relative to the expression in tumor cells grown alone (A). Three head and neck squamous cell carcinoma cell lines were grown in spheroids alone or co-cultured with their patient-matched cancer-associated fibroblasts (CAFs) for 5 days. RNA was isolated from tumor cells (separated from CAFs by magnetic activated cell sorting) and the mRNA expression of the MMP1, MMP9, POSTN, GREM1, COL1A2, FMOD, DCN, THBS1, VCAN, CTSK, and IVL genes, which, by microarray analysis, were identified as differentially expressed in co-cultures, was analyzed by qRT-PCR. The log₂ mRNA levels of co-cultured tumor cells are shown relative to the expression in tumor cells grown alone (B–D). Data were normalized to cells cultured without CAFs for each gene (mean values ± SD; n = 3). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 according to Student’s t-test.