L asparaginase

A total of 2×10⁴ cells/well seeded in 96-well plates were cultured in 90 µl RPMI-1640 medium supplemented as aforementioned (Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 12 h and then treated with 10 µl doxorubicin, cytarabine, cisplatin, gemcitabine and L-asparaginase (all Sigma-Aldrich; Merck KGaA) separately in each well with a concentration gradient for 48 h. Cell Counting Kit (CCK)-8 solution (20 µl, 5 mg/ml; Dojindo Molecular Technologies, Inc.) was used to detect the activity of cells according to the manufacturer's instructions. The absorbance was measured at 450 nm using a Multiskan FC microplate reader (Thermo Fisher Scientific, Inc.). The inhibition rate (%) was calculated as follows: (OD_control-OD_treatment/OD_control) ×100. Independent experiments were repeated at least three times.

Animal experiments were approved by the experimental animal ethics committee of Fudan University. NK-92 MI cells were stably transfected with luciferase gene and named NK-92 MI Luc. NK-92 MI or NK-92 MI Luc cells were mixed with high concentration Matrigel (Corning) and injected subcutaneously into the right flank regions of 8-week-old female NOG mice (Vital River Laboratory Animal Technology), 5 × 10⁶ cells per mouse. When the tumor volume reached 300 mm³, the mice were randomized into different groups and treated at the indicated days with vehicle, or ICT and/or GELOX regimen. ICT, gemcitabine (CSN Pharm), and oxaliplatin (CSN Pharm) were prepared in 5% DMSO and 40% polyethylene glycol 400 (CSNpharm), 5% Tween-80 (CSNpharm), and 50% PBS. L-asparaginase (Sigma) was prepared in PBS. The tumor length and width and mice weight were measured every other day. The tumor volume (mm³) was calculated using the following formula: V = 0.52 × length × width². Bioluminescence imaging was performed using IVIS (PerkinElmer) imaging system. Live animal images were acquired 10 min after intraperitoneal injection of D-Luciferin (3 mg/mouse; Beyotime). The mice were euthanized and tumor tissues were collected and weighed after treatment finished.

GH3 cells were plated at 200 cells/well in 6-well plates in complete F-12K, in the absence or presence of Gln or 1 U/mL L-asparaginase (Sigma, A3809) and 1 mM L-methionine sulfoximine (Sigma, M5379) as indicated. After 14 days, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Visible colonies were manually counted.