Xenograft Tongue Tumor Model in NSG Mice

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Augusta University. Six-week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). An orthotopic xenograft tongue tumor model was generated as we previously described [16 (link)]. Briefly, 1 × 10⁵ luciferase containing HN12 cells carrying different ATAD3A gene modifications were suspended in 50 μl of PBS/Matrigel (3:1) and injected into the anterior ~ 1/3 tongue of NSG mice under anesthesia. Twenty-one days after cell implantation, mice were imaged for bioluminescent luciferase signal using a Xenogen IVIS-200 In Vivo Imaging System (PerkinElmer, Waltham, MA). For drug treatment, 7 days after receiving ATAD3A KO or parental HN12 cells, tumor-bearing NSG mice were treated with salirasib intraperitoneally once every other day for two consecutive weeks at 30 mg/kg body weight. The mice treated with an equal volume of vehicle (PBS) were used as control. When the experiment was terminated, the primary xenografts were excised and processed for standard histological analysis with H&E staining and IHC with antibodies against p-ERK1/2 and Ki67 as we previously described [29 (link), 30 (link)].