| Objective:China is a high-risk area of esophageal carcinoma in the world and 90%type of esophageal carcinoma is esophageal squamous cell carcinoma(ESCC),The prognostic survival rate of patients with esophageal carcinoma is lower.However,cancer stem cells(CSCs)are closely related to replase,development,metastasis and drug resistance of tumors,which may be one of the causes leading to failure of cancer therapy.Interleukin 24(IL-24)has specific anti tumor activity,and recombinant IL-24 adenovirus(Ad.mda-7)can inhibit the proliferation of CSC in breast cancer with minimal toxicity to normal stem cells.Compared with recombinant IL-24 adenovirus,recombinant human IL-24 protein(rhIL-24)has the advantages with regard to tumor treatment,such as lower cytotoxicity,higher activity and specificity,and easier usage.Thus we planed to clarify the inhibitory effect of rhIL-24 on the proliferation of CSC population in esophageal squamous cell carcinoma cells in vivo,and to provide an experimental basis for the development of rhIL-24 as a biological agent for the clinical treatment of esophageal carcinoma.Methods:First,the presence of CSC in ESCC cell line KYSE450 was detected by Hoechst33342 staining.Then the CSC subpopulation in KYSE450 cells was enriched by spheroid culture in serum-free medium;The expression levels of CSC markers(CD44,CD 133,CD90 and CD271)in spheroid cells were identified by immunofluorescence staining and flow cytometry.The tumorigenic ability of the cells was identified by extreme limiting dilution assay.Secondly,KYSE450-CSC was injected subcutaneously into nude mice and treated with the engineered cell line FCHO/IL-24 secreting rhIL-24 to analyze the effect of rhIL-24 on the proliferation of KYSE450-CSC in vivo.In order to explore its possible mechanism,the expression of IL-24 receptor(IL-20R1,IL-20R2 and IL-22R1)on KYSE450 cells was detected by immunofluorescence staining.Finally,tissue samples of patients with esophageal carcinoma were collected,and the expression levels of CSC markers(CD44,CD133,CD90 and CD271)and IL-24 were detected by immunohistochemistry.The correlation between CSC marker expression levels and clinical information,and IL-24 expression level were analyzed.Results:(1)There were side population cells accounted for 0.5%in the esophageal squamous cell carcinoma cells KYSE450 by flow cytometry,which could pump out Hoechst 33342.(2)The CSC in KYSE450 cell were enriched by spheroid culture in serum-free medium.Flow cytometry results showed that KYSE450 spheroid cells expressed higher CSC markers than KYSE450 adherent cell,such as CD44(**p<0.01),CD 133(*p<0.05),CD90 and CD271(*p<0.05).(3)The tumor initiation frequency of KYSE450 spheroid cells(1/3523)was much higher than that of adherent cultured parental cells(1/62348)(***p<0.001).(4)The engineered cell line FCHO/IL-24 secreting rhIL-24 can inhibit the proliferation of subcutaneous xenografts of KYSE450 spheroid cells in nude mice.(5)Immunofluorescence staining showed that IL-24 receptor IL-20R1,IL-20R2 and IL-22R1 were expressed on the cytomembrane of KYSE450 cells.(6)CD271 and CD44 were highly expressed in tissue samples of patients with esophageal squamous cell carcinoma.High expression of CD 133 was positively correlated with pathological stage of patients(*p<0.05),and negatively correlated with survival time of patients(**p<0.05).(7)The expression levels of IL-24 and CD 133 were positively correlated in esophageal squamous cell carcinoma(*p<0.05).Conclusions:FCHO/IL-24,an engineered cell line secreting rhIL-24,could inhibit the proliferation of cancer stem cells in the esophageal squamous carcinoma cell line KYSE450 enriched by spheroid culture.The expression level of CD133 in esophageal cancer tissues may be a potential indicator for the prognosis of patients with esophageal carcinoma. |
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