Human ESCC tumor fragments were obtained from three male ESCC patients who underwent esophagectomy at the First Affiliated Hospital of Zhengzhou University (Zhengzhou, Henan, China) without any neo-adjuvant therapy (Supplementary Table 1). In China, ESCC is much more common in males than in females. According to the epidemiological investigation of ESCC incidence in 2003, ESCC incidence in males is 19.68/100,000 compared to 9.85/100,000 in females. Thus for these initial studies, we used tumor fragments from male patients only, but will examine tumors from female patients in future studies. A frozen section was stained with H&E and evaluated to confirm the diagnosis. A fresh ESCC tissue fragment was collected and transferred at 4 °C in FBS-free RPMI-1640 medium with antibiotics. Within 2 h of surgical resection, the tumor tissue was trimmed, cut into 3-5 mm pieces and implanted subcutaneously in anesthetized 6–8 week old female C.B-17 severe combined immunodeficient mice (Vital River Laboratories Co., Ltd., Beijing, China). Once mass formation reached about 1500 mm3, mice of this first generation of xenografts (named G1) were sacrificed and the tumors were passaged and expanded for two more generations (named G2 and G3). When G3 tumors reached an average volume of 250 mm3, mice were divided into three groups (seven mice per group) randomly and treated with vehicle or 2% (v/v) or 4% (v/v) DFMO in drinking water, respectively. The concentration of DFMO (2% v/v in drinking water) was founded on a previous report.13 Based on average mouse daily water intake (4 ml per day), 80 mg (4 g/kg body weight) DFMO would be consumed by a mouse per day. At the same time, we also set a higher concentration group (4%) for comparison. Tumor volume and body weight were measured every three days. Tumor volume was calculated from measurements of three diameters of the individual tumors using the following formula: tumor volume (mm3) = (length × width × height × 0.52). Mice were monitored until tumors reached 1000 mm3 total volume, at which time mice were euthanized and tumors extracted. Seven animals per group were recruited to achieve statistical significance. Mice were randomly grouped and treated without investigator blinding.