| Background and objectivesLung cancer,one of the most frequent tumor entities,is the leading cause of cancer-related mortality worldwide,with nearly 1.6 million deaths each year[7].Non-small cell lung cancer（NSCLC）,which includes adenocarcinoma,squamous cell carcinoma,large cell carcinoma,and bronchioloalveolar carcinoma,accounts for approximately 85%among all lung cancer cases[8].Approximately 70%of these patients are often diagnosed at locally-advanced or metastatic stage,resulting in the high mortality rate.Despite advances of traditional therapeutic regimens in the past few decades,the prognosis remains poor for the majority of patients（five years survival rates<15%）[9].Therefore,identifying novel biomarkers for the precision diagnosis and treatment is needed to improve the prognosis of patients with NSCLC.Cancer stem cells（CSCs）have been identified as a key mechanism of tumor initiation,metastasis,recurrence and multidrug resistance[10,11].They are a small subset of self-renewed,pluripotent,immune-privileged and long-living malignant cells.CD133 has been widely confirmed as a reprehensive marker of the CSC phenotype in some solid tumors,including lung cancer[12,13].Persistent activation of highly conserved signaling pathways,such as Notch,Hedgehog,and/or Wnt pathways partially determines the stem-like properties and tumorigenicity in CSCs[14].In addition,the stem-like properties of cancer cells have a direct link to the epithelial-to-mesenchymal transition（EMT）,an essential developmental process that is often activated during cancer invasion and metastasis[15-17].The induced EMT was accompanied with the increased ability of mammospheres formation[18].Recently,a new concept of"CSC immunology"is established which highlights that the characters of CSCs are dependent on specialized immune-niches[19].Many immune factors have been substantiated to endow tumor cell with stem-like properties via distinct molecular mechanisms in cancer[20,21].However,the molecular mechanisms underlying the plasticity of CSCs the have not been completely elucidated in NSCLC.Therefore,this study aims to explore the key molecular mechanism of TME regulating NSCLC CSCs and providing novel strategies for controlling the recurrence and metastasis of NSCLC.MethodsFor immunohistochemistry and immunofluorescence staining,a total of 86 NSCLC tissues were obtained from patients failing to receive any therapeutic intervention such as chemo-and radiotherapy before operation but received four cycles of cisplatin-based chemotherapy after surgery to investigate the expression of IFN-γand further analyze the association of IFN-γwith intercellular adhesion molecule-1（ICAM1）,CD133 and clinical parameters.Flow cytometer analysis was performed to detect the main source of IFN-γin NSCLC tissues.The effects of different dose of IFN-γon the apoptosis of NSCLC cells were tested by flow cytometer analysis and western blot.The effects of different dose of IFN-γon the stemness of NSCLC cells were tested by RT-PCR,western blot and sphere forming assay was evaluated by subcutaneous xenograft mouse model in nude mice.The identification of ICAM1was used by PCR array in NSCLC cells with or without low dose of IFN-γtreatment.Immunofluorescence staining was performed to detect the co-expression of ICAM1with CD133 in NSCLC cells with or without low dose of IFN-γtreatment.Silibinin was used to inhibit the expression of ICAM1.The effects of ihhibiting ICAM1 on the stemness of NSCLC cells treated by low dose of IFN-γwere tested by RT-PCR,western blot and sphere forming assay in NOD/SCID mice.Wsetern blot was used to investigate the signaling cascades involved in the process of IFN-γpromoting the stemness of NSCLC cells.Sphere forming assay was further performed to verify the effect of inhibiting the key molecules in the signaling cascades on the stemness of NSCLC cells treated by low dose of IFN-γ（13）Results1.The flow cytometry analysis showed that CD3+CD8+T cells,CD3+CD4+T cells,NK cells and NKT cells with a relatively high production of IFN-γwhileCD3+CD4+Foxp+Treg cells and tumor cells with a relatively low production ofIFN-γin fresh tumor tissues from patients with NSCLC.2.The IHC results demonstrated that low level of IFN-γexpression in tumor-cell-located regions was strongly correlated with brain metastasis and chemoresistance,shorter OS and PFS times.3.The immunofluorescence and IHC results suggested that low level of IFN-γ expression in tumor-cell-located regions was positively correlated with CD133 expression by tumor cells in patients with NSCLC.4.Low dose of IFN-γendowed NSCLC cells higher stem-like properties.5.PCR array and flow cytometry analysis identifed the significant up-regulation of ICAM1 as the molecular mechanism underlying increased stem-like properties mediated by low dose of IFN-γ.6.The immunofluorescence results showed that the co-expression of ICAM1 with CD133 was obviously elevated in low dose of IFN-γ-treated NSCLC cells.7.Inhibition of ICAM1 by silibinin and siRNAs abolished the elevated stem-like properties induced by low dose of IFN-γ（13）8.The results of western blot and RT-PCR showed that ICAM1 promoted the stemness of NSCLC cells treated with low dose of IFN-γvia the PI3K/Akt/Notch1 signaling pathway.9.Inhibition of PI3K,Akt or Notch1 attenuated the stem-like properties of NSCLC cells treated by low dose of IFN-γ.10.Expression level of ICAM1 was significantly upregulated by CD133+tumor cells and was positively correlated with the poor prognosis in NSCLC patients.Conclusion1.Low level of IFN-γwas closely correlated with tumor stemness and poor prognosis in patents with NSCLC.2.Low level of IFN-γas a driver of the stem-like properties of NSCLC cells through the ICAM1-PI3K-Akt-Notch1 axis.Targeting ICAM1 resulted in a remarkable inhibition of stem-like properties in NSCLC cells following low dose of IFN-γtreatment.3.ICAM1 could be used as a potential marker of CSCs and a therapeutic target for NSCLC patients,particularly for those with low level of IFN-γexpression in the tumor microenvironment. |
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