| Backgroud and ObjectiveIL-33is a multifunctional cytokine in immune regulation that activates Thl cells, Th2cells, CD8+T cells and NK cells. IL-33is associated with a broad rage of diseases, including infection-related diseases, allergy and autoimmune diseases and cardiovascular diseases. Thl cells, Th2cells, CD8+T cells, NK cells and NKT cells play impotant roles in anti-tumor immunity. We presumed that IL-33may affect the growth, metastasis and prognosis by regulating the CD8+T cells, NK cells and NKT cells.This may provide a new target for anti-tumor immunotherapy. Till now, the accurate function of IL-33in cancer remains ill-defined. In this study, the effect and mechanism of IL-33on activation of immune cells and tumor growth and metastasis was determined in IL-33transgenic mice and B16-luc melanoma/Leiws lung carcinoma pulmonary metastasis models.MethodsB16-luc cells or Leiws Lung Carcinoma (LLC) tumor suspensions were inoculated into the lateral tail vein of mice to establish the pulmonary metastasis model. The pulmonary metastasis in the IL-33transgenic mice and nontransgenic (NTG) mice was determined by bioluminescence imaging, gross anatomic and pathological observation, weight of tumor-bearing lungs and survival analysis. The percentages of CD4+T cells, CD8+T cells, B cells, NK cells and Macrophages were quantitatively analyzed in the blood and spleen from healthy, B16-luc and LLC tumor-bearing IL-33transgenic mice and NTG mice by flow cytometry. The infiltrated CD8+T cells and NK cells in the LLC pulmonary metastases from IL-33transgenic mice and NTG mice were observed by immunofluorescence staining. The CD8+T cells and NK cells were sorted from the spleen of healthy and B16-luc tumor-bearing IL-33transgenic mice, then the cytotoxicity of CD8+T cells and NK cells were detected by a4-h LDH Non-Radioactive Cytotoxicity Assay against B16-luc target cells at four different E:T ratio. The CD8+T cells and NK cells from the spleen of healthy C57BL/6J wild-type (WT) mice were treated with or without recombinant IL-33(100ng/mL), and the cytotoxicity of CD8+T and NK cells were detected by a4-h LDH Non-Radioactive Cytotoxicity Assay against B16-luc target cells. The CD8+T cells or NK cells were depleted using anti-CD8or anti-asialo GM1antibody in vivo, then the B16-luc pulmonary metastases in B16-luc tumor-bearing mice with or without depletion of CD8+T cells or NK cells were detected by bioluminescence imaging and quantified by normalized photon flux.The CD8+T cells and NK cells were sorted from the spleen of B16-luc tumor-bearing mice, and then the phosphorylation of NF-κB and expression of CD69were analyzed by western blotting in the B16-luc tumor-bearing mice. The CD8+T cells and NK cells from healthy WT mice were treated with or without recombinant mIL-33for30minutes at a dose of100ng/mL in vitro and then the phosphorylation of NF-κB and expression of CD69were analyzed by western blotting.ResultsThe results of the experiments in vivo and in vitro supported that:(1) Transgenic over-expression of IL-33attenuated tumor metastasis in the B16-luc melanoma and Lewis lung carcinoma (LLC) metastatic models. It indicated that IL-33could inhibit tumor pulmonary metastasis in vivo.(2) The percentages of CD8+T cells and NK cells in the blood and spleen and their infiltration into the pulmonary metastases were significantly increased by the transgenic over-expression of IL-33in tumor-bearing mice. It indicated that IL-33could promote the proliferation of CD8+T cells and NK cells and their infiltration into the pulmonary metastases.(3) The cytotoxicity of CD8+T and NK cells to the target B16-luc melanoma cells significantly increased in the healthy and tumor-bearing IL-33transgenic mice. Treatment with recombinant IL-33could also increase the cytotoxicity of CD8+T cells and NK cells in vitro.These results indicated that IL-33could activated CD8+T cells and NK cells in the anti-tumor immune response.(4) In addition, depletion of CD8+T cells and NK cells using anti-CD8or anti-asialo GM1antibody abolished the pulmonary metastasis inhibition mediated by IL-33. It indicated that the anti-tumor function of IL-33was mediated by the activation of CD8+T cells and NK cells.(5) IL-33stimulated the activation of NF-κB and increased CD69expression, which is a marker of the activated form of the two cell subsets, in CD8+T cells and NK cells in vivo and in vitro. It indicated that NF-κB signaling played important roles in the activation of CD8+T cells and NK cells mediated by IL-33.ConclusionsTaken togerther, our results indicated that:(1) IL-33could inhibit tumor growth and pulmonary metastasis via promoting the proliferation and activation of CD8+T cells and NK cells.(2) IL-33stimulated NF-κB signaling and promoted the proliferation, activation and infiltration into the tumor metastases of CD8+T cells and NK cells.Our results suggested that IL-33could siganificantly inhibit tumor metastasis and it may be a potential immunocytokine in anti-tumor immunotherapy. Backgroud and ObjectiveAdenocarcinoma of the lung is the most common form of lung cancer, but the cell of origin and the stages of progression of this tumor type are not well understood. It is important to establish a model of pulmonary adenocarcinoma in mice which could highly mimic the tumor initiation and progression in human. Recently, a new sporadic pulmonary tumor mouse model was established. In this model, the expression of K-ras G12D in the lung was induced by a recombinant adenovirus expressing Cre recombinase (Adeno-Cre), however, the utilization of recombinant adenovirus was unstable and transient. The aim of this study is to establish a chronic spontaneous pulmonary tumor mouse model.MethodsSPC-CRE transgenic mice were generated that express lung-specific Cre recombinant enzyme with low level. The SPC-CRE transgenic mice were mated with LSL K-ras G12D transgenic mice to produce SPC-CRE-Kras double transgenic mice. The4,5,7and9month-old SPC-Cre-Kras double transgenic mice were sacrificed and the lungs were extracted, fixed, embedded in paraffin and sliced. Hematoxylin-eosin (HE) staining was performed and observed under light microscope. Micro-CT was used to test the pulmonary nodules of SPC-CRE-Kras double transgenic mice.ResultsThe SPC-CRE-Kras double transgenic mice were generated. The expression of K-ras G12D in the SPC-CRE-Kras double transgenic mice could be induced by the Cre/Loxp recombinant enzyme system. Mild pulmonary inflammation could be found in the4month-old SPC-CRE-Kras double transgenic mice. The sporadic adenoma could be found in the lung of5month-old mice, with100%of the mice developing pulmonary adenoma (female6/6, male6/6). The size and progression of the adenoma is time dependent. The pulmonary nodules could be determined by micro-CT in the9month-old.Conclusions A SPC-CRE-Kras double transgenic chronic spontaneous pulmonary tumor mouse model was established and the expression of K-ras G12D in the SPC-CRE-Kras double transgenic mice was induced by the Cre/Loxp recombinant enzyme system. Longer progressive period from inflammation to adenoma will provide enough time for the investigation of pulmonary tumorigenesis. |
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