Development of orthotopic tumor xenograft models of esophageal squamous cell carcinoma for preclinical drug testing

Abstract

Esophageal squamous cell carcinoma (ESCC) is the predominant type of esophageal cancer mostly affecting the upper region of the esophagus. Different animal models for ESCC research have been developed, however each of them associate with their own limitations. In this study, I have developed a new upper region orthotopic tumor xenograft model in mice by injecting ESCC cells directly into the cervical esophageal wall after opening the skin and muscle layer of the mice. Mice bearing these orthotopic tumors presented similar symptoms as in the clinical situation. Biochemical analyses of the developed tumors using hematoxylin and eosin staining and immunohistochemistry revealed similar tumor features as the patient tumors by the presence of tumor invasion and angiogenesis. In addition, the procedure of establishing the orthotopic tumors is technically more effective when compared to the currently available methods. I have employed the use of this new model to test the anticancer effect of new compound, curcumin analogues. Novel curcumin analogues (SSC-3, SSC-5, SSC-6, SSC-8, SSC-9, SSC-10, SSC-19, and SSC-20) were synthesized by modifying the parental curcumin compound. Among these curcumin analogues, SSC-5 and SSC-19 exhibited most potent growth inhibitory effects on five ESCC cell lines SLMT-1, HKESC-1, HKESC-2, KYSE-270, and KYSE-450 by MTT assays (IC50 value between 0.5 and 1.5 μM). Treatment of SLMT-1 and KYSE-450 cells with SSC-5 and SSC-19 for 24 hours resulted in cell cycle arrest at G2/M phase. In KYSE-450 cell line, G2/M phase arrest (>40%, p<0.01) and sub-G1 phase (>5%, p<0.01) indicated an inhibition of cell growth and an induction of apoptosis. Western blot results also revealed a significantly higher level of apoptotic molecule cleaved-PARP (26 kDa) in SLMT-1 and KYSE-450 cells after treatment with SSC-5 and SSC-19. An attenuation of phosphorylated Bcl-2 was also observed in KYSE-450 cells. In my newly developed orthotopic tumor xenograft model developed using SLMT-1 cells, SSC-5 treatment group had the lowest percentage of mice bearing invasive SLMT-1 tumors when compared to the control experimental groups. Importantly, a significant tumor growth inhibition was observed in SSC-5 treatment group when the anticancer effect associated with such treatment was examined using my newly developed orthotopic tumor xenograft model. In summary, the newly developed orthotopic tumor xenograft model is a technically feasible and reliable model for preclinical compound testing for ESCC.