Collagenase type 2 and 4

Tumor tissues from the tumor front, matched inner tumor tissues, and matched adjacent normal lung tissues were washed three times with sterile phosphate-buffered saline (PBS). The tissues were minced with sterile scissors in a culture dish and digested for 30 min at 37°C in digestion buffer containing 0.25% collagenase type II and IV (Worthington Biochemical Corporation, Lakewood, NJ, USA). The cell suspension was washed with DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin (DMEM-S) at 500 × g for 10 min. After washing, the pellet was resuspended in fresh DMEM-S and plated onto a 25-cm 2 tissue culture flask at 37°C in a 5% CO 2 atmosphere for 2 days. Fibroblast-like adherent cells were maintained in DMEM-S. The third-or fourthpassage primary fibroblasts were used in all experiments. The primary fibroblasts isolated from the tumor front, inner tumor, and adjacent normal lung tissue were identified as F-CAFs, In-CAFs, and NFs, respectively.