| Aims:Cell-in-Cell(CIC)structure is the fourth type of cell death following cell necrosis,apoptosis,and autophagy.The CIC in some tumor tissues is positively correlated with the degree of tumor malignancy and may be a prognosis marker for cancer patients.Interleukin 24(IL-24)has a broad-spectrum and specific anti-tumor activity.It inhibits tumor proliferation by inducing tumor cell apoptosis and autophagy,and has almost no toxic effects on normal cells.In order to identify the CIC structure in esophageal carcinoma and relationship between IL-24 bioactivity and CIC,this study not only detected the CIC structure in esophageal carcinoma tissue samples,but also analyzed the effects of IL-24 on CIC of esophageal squamous cell carcinoma cells in vivo and in vitro.Methods:First,75 paired esophageal cancer patient samples(tumor tissue,paracancerous tissue,and normal tissue)were collected and detected by EML method(E-cadherin MacrophageLeukocyte;E-cadherin,CD45,and CD68 were used to label epithelial cells,leukocyte and macrophages,respectively).The correlation between the CIC density and the clinical information of esophageal carcinoma patients,and the correlation between the expression level of IL-24 protein and CIC density were analyzed.Next,nude mouse xenograft models of four different esophageal squamous cell carcinoma cells(KYSE140,KYSE150,KYSE450,and KYSE510)were prepared.After treatment with FCHO/IL-24(a stable site-specific-integrated cell line secreted rhIL-24)and FCHO cells(control cell line),the CIC in the xenograft tumor tissue samples was detected with the EML method,and the effect of cell line FCHO/IL-24 on the CIC density of the xenograft tumor tissue samples was analyzed.The IL-24 secreted by FCHO/IL-24 cells was purified by affinity chromatography.The CIC density KYSE150 and KYSE450 cells treated by rhIL-24 was analyzed by staining with phalloidin and DAPI.Results:(1)There were four subtypes of CIC in esophageal carcinoma tumor tissue,such as tumor-in-tumor(CD45-/CD68),leukocyte-in-tumor(CD45+/CD68-),tumor-in-macrophage(CD45-/CD68+)and leukocyte-in-macrophage(CD45+/CD68+).(2)The density of CIC in esophageal carcinoma paired tissue specimens was different.There were CIC structure in 54tumor tissue and the positive percentage was 68%,but noCIC in all 38 peritumored tissues and 35 normal tissues.(3)There is no statistically significant correlation between the CIC density of the homologous CIC(tumor-in-tumor)in esophageal cancer tissue and the age,sex,tissue type,lesion location,pathological grade,tumor stage,tumor size,metastasis lymph node and IL-24 expression levels,but negative correlation with the degree of differentiation of esophageal squamous cell carcinoma(p = 0.01).(4)There was a significant difference in the survival time of patients with different CIC density,such as tumor-in-tumor,leukocyte-in-tumor and total CIC,the p values were 0.0016,0.015 and 0.0089 respectively.The patients without CIC structure have the high survival rate than those with CIC structure.Namely these three subtypesCIC were negatively related with prognosis.(5)IL-24 expression level in esophageal cancer tissue samples was positively correlate with densities of total heterologous CIC(p=0.005).(6)The cell line secreted rhIL-24 not only inhibited the proliferation of the four esophageal squamous cell carcinoma cells KYSE140,KYSE150,KYSE450,and KYSE510 in vivo,but also reduced CIC density of tumor-in-tumorin tumor tissues of KYSE140 and KYSE450 cells in nude mice,as well as increased KYSE510.The CIC in tumor tissue in nude mice of cell KYSE150 was not detected.(7)The rhIL-24(5 ng/ml and 20 ng/ml)can inhibit the proliferation of KYSE150 and KYSE450 cells in vitro and reduce the CIC density of tumor-in-tumor.Conclusion:In esophageal carcinoma tumor tissue samples,IL-24 expression level was negatively related with the density of total heterologous cell-in-cell,which identified that IL-24 may promote cell-in-cell through immune system in order to inhibit tumor growth. |
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