Mkn28

Total 180 gastric tissues including 100 primary carcinomas, 4 adenomas, 6 hamartomas, 6 hyperplastic polyps, and 64 normal gastric tissues were obtained were obtained from 100 gastric cancer patients and 80 noncancer patients by surgical resection in the Kyung Hee University Medical Center (Seoul, Korea). Signed informed consent was obtained from each patient. Tissue specimens were snap-frozen in liquid N₂ and stored at −70 °C until used. Tissue slices were subjected to histopathological review and tumor specimens composed of at least 70% carcinoma cells and adjacent tissues found not to contain tumor cells were chosen for molecular analysis. Fourteen human gastric cancer cell lines (SNU5, SNU16, SNU216, SNU484, SNU601, SNU620, SNU638, SNU719, MKN1, MKN28, MKN45, MKN74, AGS, and KATO-III) were obtained from Korea Cell Line Bank (Seoul, Korea) or American Type Culture Collection (Rockville, MD).

Gastric cancer cell lines KATO-III (KCLB No. 30103) and MKN28 (KCLB No. 80102) were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were maintained in RPMI-1640 (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS; Equitech-Bio, Ingram, TX) supplemented with 100 U/ml penicillin G and 100 μg/ml streptomycin (Invitrogen). Cells were incubated at 37 °C in a humidified atmosphere containing 20% O₂ and 5% CO₂. Type I collagen extracted from rat tail (BD Biosciences, Franklin Lakes, NJ) was dissolved in 0.02 N acetic acid and used for coating tissue culture dishes and inserts for migration assays (5 μg/cm²). Collagen-coated dishes and inserts were washed with PBS immediately before use.
DDR1 inhibitor 3-(2-(pyrazolo[1,5-a]pyrimidin-6-yl)ethynyl)benzamide (7rh benzamide) was dissolved in DMSO to a final stock concentration of 2.5 mg/ml as previously reported [27 (link)].

To validate antibodies for NGF and HO1 used in immunohistochemical staining, we performed western blotting analysis. Four human gastric cancer cell lines (MKN28, MKN45, KATO-III, and NCI-N87) were purchased from the Korean Cell line Bank (KCLB, Seoul, Korea) and cultured in RPMI1640 media. The cells were lysed with PRO-PREP Protein Extraction Solution (iNtRON Biotechnology Inc., Seongnam, Korea). Western blot was performed with primary antibodies for the NGF (Abcam, Cambridge, UK), HO1 (Enzo Life Sciences, Farmingdale, USA), and actin (Sigma, St. Louis, USA).

Twelve gastric cancer cell lines (six were established from male patients: NCI-N87, SNU-484, SNU-1, SNU-638, KATOⅢ, MKN-1; six were established from female patients: SNU-5, MKN-28, SNU-216, SNU-16, AGS, SNU-620) were obtained from Korean Cell Line Bank (KCLB; Seoul, Korea). All of the cells were cultured in RPMI 1640 (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin; Gibco BRL).

The human GC cell lines NCI-N87, SNU16, MKN7, MKN28, and AGS were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea) and maintained in RPMI-1640 supplemented with 10% fetal bovine serum. The cells were cultured at 37 °C with 100% humidity and 5% CO₂. LOXO-101, entrectinib, dovitinib, dovitinib lactate, dovitinib dilactic acid, regorafenib, cabozantinib, and crizotinib were purchased from Selleck Chemicals (Houston, TX, USA).

The gastric cancer cell line AGS was obtained from the American Type Culture Collection (Manassas, VA, USA). EBV (−) GC cell lines (SNU-601, MKN-1, and MKN-28) and EBV (+) GC cell lines (SNU-719 and NCC-24) were obtained from the Korean Cell Line Bank (Seoul, Korea). The six cell lines were cultured in RPMI1640 medium. The EBV (+) YCCEL1 GC cell line was a kind gift from Dr. SY Rha from the Yonsei University College of Medicine. YCCEL1 cells were cultured in minimum essential medium (MEM). Media were supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA). Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Informed consent was obtained from all patients and the experimental protocol was reviewed and approved by the Tissue Ethical Committee of Korea University Ansan Hospital for the use of tissue specimens (No. 2016-004). The method was carried out in accordance with the committee’s approved guidelines. Paraffin-embedded GC and non-malignant gastric tissue specimens were used for immunohistochemistry.