Based on the in vitro assay results, we investigated the function of ODC in ESCC tumorigenesis in an athymic nude xenograft mouse model. Mice were divided into three groups and inoculated in the right flank with shMock-infected, shODC#2-infected, or shODC#4-infected KYSE450 cells (3 × 106 cells per mouse, 4 mice per group). The body weight of mice increased normally with growth for the duration of the study, indicating no adverse effects from the respective inoculation (Fig. 4a). At 56 days after inoculation, when the tumor volume of the shMock group reached 1000 mm3, all experimental mice were euthanized and tumors extracted for IHC analysis (Supplementary Fig. 2). Our data showed that shMock-infected KYSE450 cells formed markedly larger xenograft tumors in nude mice compared to the shODC-infected groups (p < 0.01, Fig. 4b). IHC results showed that expression of ODC, Ki-67 and PCNA proliferation markers was attenuated. In addition, expression of the anti-apoptosis marker Bcl-2 was also decreased, whereas expression of cleaved caspase 3 was increased (Fig. 4c). These results confirmed that inhibiting ODC expression in ESCC cells suppresses proliferation and induces apoptosis, leading to attenuation of ESCC tumorigenesis.Fig. 4shODC suppresses the tumor-forming ability of ESCC cells. a After inoculation, the body weights of all mice remained stably increased. b shODC significantly suppresses KYSE450 xenograft tumor volume compared with the shMock group. c IHC analysis was performed to determine the expression levels of ODC, PCNA, Ki-67, cleaved caspase 3, and Bcl-2 in ESCC xenograft tumors expressing shMock or shODC. 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