Murine xenograft models

Male athymic nude mice (4–6 weeks old) were maintained as described [33]. All studies were approved by the Institutional Animal Care and Use Committee at Vanderbilt University. SK-N-AS and BE(2)-C cells xenografts were established as previously described [33]. Briefly, 1 × 10⁶ cells/100 μL of HBSS were injected subcutaneously into flanks using a 26-gauge needle (n = 10 per group). Mice were monitored daily for xenograft formation and assessed by measuring the two greatest perpendicular tumor diameter with vernier calipers (Mitutoyo, Aurora, IL). Xenograft volumes were estimated using the following formula [(length × width²)/2]. Weight and tumor volume were recorded biweekly. At four weeks of post-injection, mice were euthanized when they met the institutional euthanasia criteria for tumor size and overall health condition. The tumors were excised, weighed, and fixed in 10% buffered formalin. Tumor tissues were further processed for embedding in paraffin, sectioned and stained with hematoxylin and eosin at Translational Pathology Shared Resource Laboratory in Vanderbilt University Medical Center.