In vivo xenograft tumor model of human NSCLC

A549 cells (1 × 10⁶ cells in 100 μL) were injected subcutaneously under the right axilla of the mice. Tumor volume was monitored by measuring the two maximum perpendicular tumor diameters with vernier caliper every other day. All tumor-bearing mice were randomly divided into four groups: the control group, the aspirin group, the celecoxib group and the combination group. When the tumor reached about 100–150 mm³ on the eighth day, the treatment was initiated. Aspirin (100 mg/kg body weight) was dissolved in PBS and used as daily drinking water for mice in the aspirin group or the combination group. The mice in the celecoxib group or the combination group were injected intraperitoneally (i.p.) with celecoxib (50 mg/kg body weight) dissolved in 100% DMSO every other day. Control mice were given sterile water daily and received i.p. injection of DMSO for the same period of time as the drug treatment groups. The drug treatment cycle was 28 days. Mice were weighed every two days and the maximum vertical length of all measurable tumors was measured using a vernier caliper every other day. Anti-tumor activity of treatments was evaluated by tumor growth inhibition. The formula, tumor volume = length × width² × 0.52 was used to mimic the tumor volume. At the end of the study, the tumors were collected and weighed. In a parallel animal assay (totally four groups, and six mice per group), the tumor establishment and drug treatment are the same as described previously. On the 28th day, mice were euthanized. Tumors were collected, fixed with 4% paraformaldehyde, embedded in paraffin and sectioned for hematoxylin-eosin (HE) staining according to standard histological procedures.²⁴ Apoptotic cells in tumor sections (two sections per mouse, four mice in total) were visualized by the TUNEL technique and further verified by immunohistochemistry using anti-cleaved caspase-3.