The Role And Mechanism Of Tumor-educated Lung Epithelial Cells In Promoting The Formation Of Pre-metastatic Niche

Tumor metastasis is the main cause of tumor death.And the mechanism of its occurrence and development is a hot but difficult topic in oncology.Recently,the concept of pre-metastatic niche has uncovered the relationship between metastatic tumor cells and distant organ microenvironment.Only few tumor cells entering the circulatory system can colonize and grow in distant organs,and most of them will be destined to apoptosis,so the"soil"in the metastatic organs is important for tumor cells to settle down.Bone marrow-derived hematopoietic progenitor cells expressing vascular endothelial growth factor receptor 1(VEGFR1)can be recruited in metastatic organs induced by tumor-derived vascular endothelial growth factor.They can promote the expression of metastasis-related cytokines and chemokines in the target organs,thus providing a suitable microenvironment for subsequent tumor metastasis.Studies have shown that a variety of tumor-derived secreted factors(TDSFs)and cell components in the target organs are involved in the formation of the pre-metastatic niche.Our laboratory has been engaged in the study of the mechanism of pre-metastatic niche formation,and put forward six characteristicsthatdefineit,includingimmunosuppression,inflammation,angiogenesis/vascular permeability,lymphangiogenesis,organ selectivity,and metabolism,epigenetic regulation,and stromal remodeling.However,the mechanism of pre-metastatic niche and the complex interaction between tumor cells and host microenvironment are still not clear.Lung is one of the common organs of tumor metastasis,which attracts much attention for the pre-metastatic niche study.It's reported that lung stromal cells(including fibroblasts,endothelial cells,etc.)can recruit bone marrow-derived cells(BMDCs)by secreting chemokines,cytokines and vesicles.They can promote tumor angiogenesis,stromal remodeling,immunosuppressive microenvironment formation,enhance the colonization of circulating tumor cells(CTC),and further promote the occurrence of tumor lung metastasis.As the main component cells of alveoli,lung epithelial cells can recognize pathogenic and danger-associatedsignalstoinitiateinflammatoryresponse,andplayan immunomodulatory function to maintain lung homeostasis.However,abnormal inflammation caused by long-term stimulation of lung epithelial cells is also cause for chronic lung inflammation.Therefore,the immunoregulatory function of lung epithelial cells in inflammation suggests their potential function in tumor-induced inflammatory microenvironment.However,as an important component of alveoli,the function of lung epithelial cells in pre-metastatic niche has been poorly studied.A thorough study on the function of lung epithelial cells will provide new insights into tumor metastasis and organotropic metastasis,and provide new targets for the prevention and treatment of tumor metastasis.On the other hand,the important factor modulating the target organ microenvironment is TDSFs.Tumor cells can mobilize BMDCs to form inflammatory environment in pre-metastatic organs by secreting soluble molecules and vesicles,thus promoting the colonization of tumor cells.The vesicles are mainly exosomes,which are vesicular bodies encapsulated by lipid bilayer membrane with 30-100 nm diameters and play an important role in intercellular substance and information exchange.Studies have shown that the tumor-derived exosomes contains cytokines,miRNA,lncRNA and so on,which can promote the alternation of immune cell function in the pre-metastatic niche.These studies provide a new perspective for exploring the functional remodeling of stromal cells in metastatic organs and the formation of pre-metastatic niche.Based on the above research,in order to explore the functional changes of tumor-educated lung epithelial cells,we used a mouse model of spontaneous metastasis of breast cancer in situ.Then we isolated and sequenced the lung epithelial cells to analyze the functional changes of lung epithelial cells systematically.From in vitro coculture study,it was found that tumor-educated lung epithelial cells could inhibit the proliferation of CD4~+T cells and induce the production of regulatory T cells.The mechanism study showed that the tumors can increase the expression of transcription factor Zeb2 in lung epithelial cells via exosomes,and promote the secretion of IL-10 in lung epithelial cells,thus exerting the immunosuppressive function.Our study uncovered the new function and mechanism for tumor-educated lung epithelial cells to promote the formation of pre-metastatic niche,thus providing new ideas for tumor treatment and early intervention of tumor metastasis.Part ? Tumor-educated lung epithelial cells inhibit T cell function in pre-metastatic nicheObjective:To investigate the functional changes of tumor-educated lung epithelial cells and its effect on the function of T cells.Methods:1.We established spontaneous lung metastasis model of breast cancer in situ in mice and sequenced the transcriptome of lung epithelial cells in the pre-metastatic niche.2.Lung epithelial cells from tumor-bearing or wild-type mice were co-cultured with various immune cells in vitro and verified in vivo by flow cytometry analysis in MMTV/PyMT mice model.3.We studied the effect of tumor-educated lung epithelial cells on the proliferation of CD4~+T cells and the induction of Treg in vitro.Results:1.The expression of anti-inflammatory molecules in lung epithelial cells of tumor-bearing mice was up-regulated compared with that of wild-type mice,while the expression of pro-inflammatory related molecules was down-regulated.2.Compared with wild-type mice,the number of CD4~+T cells decreased and the proportion of Treg significantly increased after co-cultured with tumor-educated lung epithelial cells.3.The proportion of CD3~+T cells,NK cells and DC cells in the lungs of MMTV/PyMT mice was lower than that of wild-type mice,while the proportion of Treg and neutrophils was significantly increased.4.Tumor-educated lung epithelial cells can significantly inhibit the proliferation of CD4~+T cells and induce the production of Treg.Conclusion:Tumor cells can induce the functional changes of lung epithelial cells.It can significantly inhibit the proliferation of CD4~+T cells and induce the production of Treg.Part ? Mechanism of tumor-educated pulmonary epithelial cells inhibiting T cell function in pre-metastatic nicheObjective:To investigate the mechanism of the immunosuppressive function of tumor-educated lung epithelial cells.Method:1.We detected the proliferation of CD4~+T cells by flow cytometry analysis after co-cultured with lung epithelial cells through transwell.2.We used real-time quantitative PCR to detect the mRNA expression of IL-10 in tumor-bearing and normal mouse.3.We detected IL-10 expression in lung epithelial cells of tumor-bearing mice by intracellular staining and enzyme-linked immunosorbent assay.4.Flow cytometry analysis was used to detect the proliferation of CD4~+T cells after blocking IL-10 in vitro.5.Establishment of exosome-educated model and detection of pre-metastatic gene expression by real-time fluorescence quantitative PCR.6.Establishment of tumor metastasis model and detection of lung metastasis by in vivo imaging of small animals.7.We extracted tumor-derived exosomes and labeled with membrane dye.The lung of mice educated by exosomes were made into frozen sections and the type of dye positive cells were detected by immunofluorescence.8.We used real-time quantitative PCR to detect the mRNA expression of IL-10 in MLE-12 after stimulated by tumor exosomes.9.The mRNA expression of IL-10 was detected by real-time quantitative PCR after interfering the transcription factors in mouse lung epithelial cell line MLE-12 in vitro.Results:1.Tumor-educated lung epithelial cells could still inhibit the proliferation of CD4~+T cells by co-cultured in transwell.2.The mRNA expression level of IL-10 in lung epithelial cells of tumor-bearing mice was higher than that of control mice in the model of breast cancer in situ and MMTV/PyMT mice model.3.The protein level of IL-10 in lung epithelial cells educated by primary tumor was significantly increased.4.Blocking of IL-10 in vitro can reverse the inhibitory effect of tumor-educated lung epithelial cells on the proliferation of CD4~+T cells.5.After educated by tumor exosomes,the gene expression of pre-metastatic niche S100a8,S100a9,Bv8,Mmp9 and Fibronectin increased.6.After educated by tumor exosomes,lung metastasis increased.7.Lung epithelial cells are the main cell types for uptake of tumor-derived exosomes in the lung.8.The exosomes derived from tumor tissues can significantly stimulate the mRNA expression of IL-10 in lung epithelial cells,which can be used to simulate the education of lung epithelial cells by tumor cells.9.Tumor-derived exosomes can promote the expression of transcription factor Zeb2 to induce the expression of IL-10.Conclusion:Primary tumor can promote the expression of transcription factor Zeb2 in lung epithelial cells by secreting exosomes,thus promoting the expression of IL-10 and inhibiting the proliferation of CD4~+T cells and induce the production of Treg.In summary,we studied the functional alternation of tumor-educated lung epithelial cells in the pre-metastatic niche and the underlying mechanisms.Our study clearly elucidates the negative immunomodulatory effect of tumor-educated lung epithelial cells,providing a new understanding for the role of lung epithelial cells in pre-metastatic niche formation and tumor metastasis.