The Role And Mechanism Of Myeloid Cell NFE2L1 In Tumor Metastasis

Object:Cancer is an intractable disease that affects human health.At present,human beings still cannot fully understand and overcome this intractable disease,which is the major disease threatening human life in today’s world.Moreover,the occurrence and development of cancer are closely related to chronic inflammation and oxidative stress.Nuclear factor erythroid 2-related factor 1（NFE2L1）is a core transcription factor regulating cellular oxidative stress,which provides necessary protection against carcinogenic damage induced by foreign chemicals and plays an important role in maintaining redox homeostasis in the body.As an important member of the cellular immune system of the body,macrophage is an important component of the innate immune response and adaptive immune response,and plays an important role in cell phagocytosis,antigen presentation,tissue repair and other aspects.Tumor Associated Macrophage（TAM）is a kind of a large number of infiltration of mononuclear macrophages in multiple organs,through the secretion of cytokines,growth factors and other many kinds of active medium role in the Tumor microenvironment,and the stimulation of many kinds of active factor in Tumor microenvironment,heterogeneity and plasticity,with functions involved in the regulation of Tumor development.Therefore,in this study,Nfe2l1-KI and Nfe2l1（M）-KO mice and mouse macrophage line RAW 264.7 cells were used as models to study the role of NFE2L1 in tumor metastasis and explore the possible mechanism of NFE2L1 regulating TAM,so as to provide a new target and theoretical basis for clinical tumor immunotherapy.Methods:1.Construction of Nfe2l1（M）-KO mice and identification of mouse genotype:Mice with C57BL/6 genetic background were used to construct myeloid cell specific Nfe2l1knockout mice,which were hybridized by Nfe2l1^flox/flox and mononuclear macrophage Lyz2-Cre,and have been used for routine breeding.DNA was amplified by mouse tail PCR,and the mouse DNA bands were obtained by agarose gel imaging system,and the genotypes were identified.2.Primary macrophage extraction and purity identification:After the mouse surface specific antigen was selected,the cells were ressuspended in 200μl PBS at a density of1×10⁶,and corresponding antibodies were added according to the proportion of flow antibody instructions.After incubation at room temperature for 30 minutes,the purity of macrophages was detected by flow cytometry.3.Confirmation of murine-specific knockout effect of Nfe2l1（M）-KO:The m RNA expression level of Nfe2l1 in bone-marrow induced macrophages was detected by real-time quantitative polymerase chain reaction（q PCR）to determine the knockout effect of Nfe2l1（M）-KO mice.4.Mouse subcutaneous tumor-bearing model and lung metastasis model construction:Nfe2l1-KI and Nfe2l1（M）-KO mice were subcutaneously injected with melanoma cells（1×10⁶/mouse）.Tumor size was measured every other day,and tumor tissues were collected at the end of the experiment.Nfe2l1-KI and Nfe2l1（M）-KO mice were injected with 1×10⁵ melanoma cells per mouse through tail vein,and lung tissue was collected from the mice two weeks later.5.Evaluation and identification of mouse lung metastatic model:When collecting mice,the number of lung surface tumors was measured,the volume and average of lung surface tumors were calculated,and the weight of lung and spleen was measured to evaluate tumor load.The lung tissue was embedded in sections and HE staining was used to evaluate the severity of lung metastasis.6.Wound healing experiment:the supernatant of RAW 264.7 Scramble and RAW264.7 Nfe2l1-KD cells was collected to treat the melanoma cells,and the change of wound healing of melanoma cells at 6 h,24 h and 48 h was observed,and the wound healing rate was calculated.7.Treatment of macrophages with supernatant of tumor cells:supernatant of melanoma cells was used to simulate tumor microenvironment,and the BMDM of Nfe2l1（M）-KO and Nfe2l1-KI were treated respectively.m RNA expression levels of markers of M2 macrophages,such as Il10,Vegf-αand Mmp9,were detected by RT-q PCR.Results:1.Nfe2l1-KI and Nfe2l1（M）-KO mice were successfully constructed:The model of NFE2L1 myeloid cell specific knockout mouse（Nfe2l1（M）-KO）was confirmed by genotype analysis.2.Bone marrow primary macrophages were successfully extracted and identified.The surface markers of macrophages were detected by flow cytometry,and the purity of macrophages was>80%.The expression level of Nfe2l1 in primary bone marrow macrophages of mice in Nfe2l1（M）-KO group was significantly lower than that in Nfe2l1-KI group,with statistical significance（P<0.05）.3.Subcutaneous tumor-bearing model was successfully constructed:subcutaneous injection of tumor cells was used to make the model,and the tumor growth of mice was observed.Data showed that compared with the control group,the tumor weight and size of mice in the Nfe2l1（M）-KO group were not significantly different from those in the Nfe2l1-KI group,but mice in the Nfe2l1（M）-KO group were more prone to spontaneous metastasis,suggesting that the specific deletion of Nfe2l1 in myeloid cells may have no effect on tumor growth but promote tumor metastasis.Therefore,subsequent tumor metastasis model was established.4.Successful construction of mouse lung metastasis model:In this study,mice with C57BL/6 as genetic background were used for modeling by injection of tumor cells through caudal vein,and lung tissue of mice was collected when reaching the end point of the experiment.Black tumors can be observed on the lung surface of mice after the successful establishment of the model.At the same time,Adenomatoid hyperplasia can be found in the lung of mice after the section staining of the lung tissue of mice,and the lung metastasis model of C57BL/6 mice is successfully established.5.Deletion of myeloid NFE2L1 promotes tumor metastasis:lung metastases were modeled by injection of tumor cells into a caudal vein,and body weight was monitored throughout the modeling period in both groups.The data showed that there was no significant difference in the effect of caudal vein injection of tumor cells on body weight.At the end of the experiment,according to the general photographs,it was significantly observed that the Nfe2l1（M）-KO group had more severe lung metastasis than the Nfe2l1-KI group.The lung and spleen tissues of mice were weighed and corrected by tibia length.It was found that there was no significant difference in organ coefficients between the Nfe2l1（M）-KO group and the Nfe2l1-KI group.According to tumor load assessment,the number of tumor metastases on lung surface in the Nfe2l1（M）-KO group was more than that in the Nfe2l1-KI group,and the difference was statistically significant（P<0.05）.The average diameter of lung surface tumor in mice was calculated,and it was found that the average diameter of lung surface tumor in the Nfe2l1（M）-KO group and the Nfe2l1-KI group showed no significant difference.The calculation of lung surface tumor volume showed no significant difference in tumor volume between the Nfe2l1（M）-KO group and the Nfe2l1-KI group.6.The proportion of M2 type macrophages in bone marrow macrophages increased:The results showed that there was no significant difference in the proportion of CD4⁺T cell and CD8⁺T cell in spleen tissue of mice in the Nfe2l1（M）-KO group and the Nfe2l1-KI group,but the proportion of M2 type macrophages in bone marrow macrophages increased slightly.7.Supernatant of RAW 264.7 Nfe2l1-KD can promote tumor cell migration:Melanoma cells were treated with the culture supernatant of RAW 264.7 Scramble and RAW 264.7 Nfe2l1-KD cells,and the wound healing of melanoma cells at 6,24,and 48 h were observed.The results showed that compared with the RAW 264.7 Scramble group,the RAW 264.7 Nfe2l1-KD group had a stronger wound healing ability and a faster wound healing rate.8.Tumor cells that can promote the supernatant fluid macrophages to M2 polarization:to investigate lung metastasis in mice model of Nfe2l1 regulation function of macrophage polarization,we utilize the supernatant of melanoma cells simulated tumor microenvironment,respectively applied to Nfe2l1（M）-KO and Nfe2l1-KI BMDM,RT-q PCR results showed that the m RNA expression levels of markers of M2 macrophages such as Il10,Vegf-αand Mmp9 were increased,and the difference was statistically significant（P<0.05）.Conclusion:1.Nfe2l1（M）-KO mice were successfully constructed.2.The specific deletion of NFE2L1 in myeloid cells has no significant effect on tumor growth,but can promote tumor metastasis.3.NFE2L1 may play a role in promoting tumor metastasis by promoting the polarization of macrophages into M2 macrophages.