We established 3 ESCC PDX mouse models, referred to as EG20, EG5, and EG37 (Supplementary Table 1) to test the effectiveness of DFMO. None of the patients donating tumors received neo-adjuvant therapy. EG20 tumors were implanted and when the tumors reached an average volume of 250 mm3, mice were divided into 3 groups and treated with vehicle, 2 or 4% (v/v) DFMO in drinking water, respectively. From the 1st day of treatment, body weight and tumor volume were measured every 3 days. Results indicated that over 33 days of treatment, DFMO had no effect on mouse body weight compared with the vehicle-treated group. This indicated that even the highest dose of DFMO (4%) was tolerable and safe (Fig. 6a). Beginning at day 9, the average tumor volume of each DFMO treatment group appeared to be significantly smaller than the average volume of the vehicle-treated group (Fig. 6b). After 33 days of treatment, the tumor volume of the vehicle group reached 1000 mm3 and all experimental mice were euthanized and tumors extracted and weighed. Final results showed that DFMO treatment significantly decreased PDX tumor weight compared to vehicle-treated control mice (p < 0.01, Fig. 6c). No significant difference was observed in either tumor volume or in tumor weight between the two DFMO-treated groups (Fig. 6b, c), indicating that even the lowest dose of DFMO was effective. Similar results were obtained from the other two ESCC PDX models, EG5 and EG37 (Supplementary Fig. 3). Even the low dose of DFMO treatment (2%) could significantly suppress the progression of ESCC PDX tumors. We also examined the expression of PCNA and Ki-67 proliferation markers and cleaved caspase 3 and Bcl-2 apoptosis markers by IHC. Our results showed that DFMO treatment suppressed proliferation and induced apoptosis of ESCC cells (Fig. 6d).Fig. 6DFMO inhibits ESCC progression in a PDX model. A PDX model of mice implanted with human ESCC (EG20) were divided into three groups and treated with vehicle or 2% (v/v) or 4% (v/v) DFMO in drinking water for a total of 33 days. a From the 1st day of treatment, body weight and tumor volume were measured every 3 days. DFMO did not significantly reduce mouse body weight compared with the vehicle group. b From the 9th day of treatment, the average tumor volume of both DFMO-treated groups appeared to be significantly less than the vehicle-treated group. c Compared with vehicle, DFMO treatment significantly decreased the weight of the PDX tumors. d IHC analysis was performed to determine the expression levels of PCNA, Ki-67, cleaved caspase 3, and Bcl-2 in ESCC PDX tumors treated with vehicle or DFMO. Representative photographs for each antibody and each group are shown. The integrated optical density (IOD) was evaluated using the Image-Pro Plus software (v.6.0) program. All data are shown as mean values ± S.D