| ObjectiveIL-37 is a new immunonegative regulatory molecule discovered in 2000 and belongs to the cytokine of the IL-1 family.IL-37 includes 5 spliceosomes(IL-37a-e).IL-37b is the largest subtype and is widely expressed in human organs and tissues.The current research on IL-37 mainly focuses on this subtype,found that IL-37b is involved in the control of various inflammatory diseases(such as systemic lupus erythematosus,atherosclerosis,psoriasis,acute coronary syndrome,rheumatoid arthritis,etc.),and in fibrosarcoma,liver Cell cancer,cervical cancer,breast cancer,kidney cancer,non-small cell lung cancer and other tumors play an anti-tumor effect.IL-37d has only one exon 2 less than IL-37b,which is predicted to be a functional subtype.We constructed IL-37d transgenic mice in our laboratory in the early stage and proved IL-37d in the endotoxemia model It has an anti-inflammatory effect like IL-37b,but its role in tumors is unclear.This subject uses IL-37d transgenic mice,using mouse B16/F10 melanoma cells and LLC lung cancer cells to establish subcutaneous xenograft and lung metastasis models,and studies the role of IL-37d in tumor growth and metastasis;before metastasis from tumor,the microenvironment began to study the effect of IL-37d on the invasion of neutrophils in tumor metastases and the migration of bone marrow-derived neutrophils,and the mechanism of action was initially discussed.This thesis includes four parts:1.The effect of IL-37d on the growth of subcutaneous transplanted tumors in mice;2.The effect of IL-37d on the progress of lung metastatic tumors and the survival rate of mice;3.The effect of IL-37d on tumor metastasis The effect and mechanism of neutrophil infiltration in the focus microenvironment;Fourth,the effect and mechanism of IL-37d on the spontaneous migration of tumor-induced bone marrow neutrophils.MethodsI.The effect of IL-37d on the growth of subcutaneous transplanted tumor in mice6-8 weeks of male C57BL/6 WT and IL-37d transgenic mice were inoculated with mouse B16/F10 melanoma cells on the back to establish a subcutaneous xenograft model.After the tumor grew on day 8,the mice were measured every other day.Back tumor size,when the mouse tumor reaches 100m m2,dissect the primary tumor and weigh,measure the size of the tumor,and take pictures of the appearance.To examine the effect of IL-37d on the growth of subcutaneous primary tumors.Ⅱ.The effect of IL-37d on the progression of lung metastases and the survival rate of mice(Ⅰ)In vivo imaging detection of the effect of IL-37d on the progression of lung metastasesInject 6-16-week male C57BL/6 WT and IL-37d transgenic mice into the tail veins with fluorescently labeled B16/F10 and LLC cells to construct a lung metastasis model,and use Shandong University for transformation at 1W,2W,and 3 W non-invasive bioluminescence imaging of isolated luciferase on the medical sharing platform performs live imaging of mice to detect the progress of lung tumors.After the mice died(about 3W),the cadavers were dissected to take pictures of the appearance of the lungs,weigh them,and take the mouse lung tissue sections for embedding.(Ⅱ)The effect of IL-37d on the survival rate of metastatic tumor miceB16/F10 and LLC cells were inoculated into the tail vein of male C57BL/6 WT and IL-37d transgenic mice of 6-8 weeks respectively to construct a lung metastasis model,weighed daily,observe the survival status of the mice,and record each The time of death of the mice.After the mice died,the cadavers were dissected and the appearance of the lungs was photographed,weighed,and the lung tissue sections of the mice were taken for embeddingⅢ.Effect and mechanism of IL-37d on neutrophil infiltration in tumor microenvironment(Ⅰ)PAS staining and flow cytometry to detect the effect of IL-37d on the number of neutrophils in the lungs of metastatic tumorsLLC cells were inoculated into the tail veins of male C57BL/6 WT and IL-37d transgenic mice of 6-8 weeks respectively to construct a model of lung metastasis.The mice were dissected at 1 W,the appearance of the lungs was photographed,weighed,and take a part of the lung tissue for embedding and preparation of tissue sections,perform PAS staining to observe the morphology of the tissue and the number of lung neutrophils recruited;take another part of the lung tissue to prepare a single cell suspension,labeled with PE-Cy7 anti-CD11b After staining with antibodies and PE-labeled anti-Ly6G antibodies,the number of neutrophils in the lung tissue of mice was detected by flow cytometry.(Ⅱ)Real-time quantitative PCR and immunohistochemistry to detect the effect of IL-37d on the expression of lung neutrophil-related molecules in the metastatic tumor model1.Real-time quantitative PCR method to detect the dynamic changes of lung neutrophil-related molecular expression during the progression of lung metastasesLLC cells were inoculated into the tail veins of male C57BL/6 WT and IL-37d transgenic mice of 6-8 weeks respectively to construct lung metastasis models.The mice were dissected at 1W,2W and 3W,and the lungs were photographed for appearance,weigh,extract total RNA from lung tissue and reverse transcribe into cDNA,and then use fluorescence quantitative PCR to detect S100a8,S100a9,MMP9,TLR3,CXCL1,CXCL2,CXCL5,CXCL5,CXCR2,CXCR4 and CXCR7 and IL-6,TNF-The expression of α,IL-1β in the lungs to determine the dynamic changes in the expression of neutrophil-related molecules during the formation of metastatic tumors.2.Using real-time quantitative PCR and immunohistochemistry to analyze the effect of IL-37d on the expression of neutrophil-related molecules in the early lung metastasesLLC cells were inoculated into the tail vein of male C57BL/6 WT and IL-37d transgenic mice of 6-8 weeks to construct a lung metastasis model.When the tumor was inoculated for 1 W,the mice were dissected and the lungs were photographed,Weigh and take mouse lung tissue sections for embedding,and use TLR3,S100a8/9 and MMP9 specific antibodies for immunohistochemical staining to detect the expression of TLR3,S100a8/9 and MMP9;extract mouse lung tissue RNA and detect S100a8,S100a9 and MMP9 expression.Ⅳ.The effect and mechanism of IL-37d on tumor-induced spontaneous migration of bone marrow neutrophils(1)The effect of IL-37d on the spontaneous migration of bone marrow neutrophils was detected by Transwell and flow cytometryLLC cells were inoculated into the tail veins of male C57BL/6 WT and IL-37d transgenic mice of 6-8 weeks respectively to construct lung metastasis models.Dissect the mice when the tumor is inoculated at 1W and 2W,and take bone marrow to prepare single cells After removing red blood cells,Ly6G+neutrophils were separated and purified from mouse bone marrow by immunomagnetic beads,and the purity of neutrophils was detected by flow cytometry;the extracted neutrophils were migrated by Transwell The experiment then counted the neutrophils migrating through Transwell by flow cytometry(II)Using Racl specific inhibitors to analyze whether the migration ability of bone marrow neutrophils is affected by the Racl pathwayTo explore whether L-37d affects the migration ability of bone marrow neutrophils through the Rac1 pathway,we used Rac1 inhibitors to block Rac1 activity,and then carried out migration experiments as described aboveResultsI.The effect of IL-37d on the growth of subcutaneous xenograftsWe have previously used IL-37d transgenic mice and LPS-induced endotoxemia model to study the inhibitory effect of IL-37d on inflammation,but its effect on tumor growth,metastasis and tumor microenvironment is still unclear.In order to explore the direct role of IL-37d in tumorigenesis and development,first C57BL/6 WT and IL-37d transgenic mice were inoculated with mouse B16/F10 melanoma cells on the back to prepare a subcutaneous tumorigenic model.Through tumor growth curve and tumor weight analysis,it was found that the tumor growth of IL-37d transgenic mice was slower than that of wild-type mice,and the tumor weight was lower.It is suggested that IL-37d can inhibit the growth of transplanted tumors in mice.Ⅱ.The effect of IL-37d on the progression of lung metastases and the survival rate of mice(Ⅰ)The effect of IL-37d on the progression of lung metastasesIn order to study the effect of IL-37d tumor metastasis,we used mouse melanoma cells(B1 6/F10)and lung cancer cells(LLC)labeled with luciferase,and injected tumor cells through the tail vein of mice to build a model of lung metastasis.The effects of IL-37d on the progression of lung metastases were analyzed by in vivo imaging at 1W,2W,and 3W after tumor injection.We found that in two different lung metastasis models,the fluorescence intensity of lung metastasis tumor cells in IL-37d mice was lower than that in WT mice.After three weeks of in vivo imaging,the mice were sacrificed to find wild-type lung tumors.The size and number are larger or larger than those in IL-37d group.It suggests that IL-37d can inhibit the progress of lung metastases(Ⅱ)The effect of IL-37d on the survival rate of lung metastasis miceIn order to determine the effect of IL-37d on tumor metastasis,we used tail vein injection of B16 cell LLC cells to construct a lung metastasis model,and further observed the effect of IL-37d on the survival status of metastatic tumor mice.It was found that in the metastatic tumor model of B16 cells,IL-37d could significantly increase the survival time of tumor-bearing mice.The average survival time was 29.3 days in the WT group and 36.7 days in the IL-37d group.At 35 days,WT mice all died,but one of the IL-37d group was still alive at 50 days,and the lung tumor was not obvious after dissection.In the mouse LLC cell metastasis model,IL-37 inhibited the tumor more obviously.By day 60,the survival rate of WT mice was 16.7%(1/6),while the IL-37d mice had 100%Survived(6/6).Dissected the remaining surviving mice,2 surviving WT mice,although there were no obvious metastases in the lungs,metastases were found in the skin and kidney;3 out of 6 surviving IL-37d mice had obvious lung The metastases were visible to the naked eye,and the other three had no obvious metastases.These results indicate that IL-37d can significantly improve the survival time and survival rate of lung metastasis miceⅢ.The effect and mechanism of IL-37d on neutrophil infiltration in the microenvironment of tumor metastases(Ⅰ)The effect of IL-37d on the neutrophil infiltration in the lungs of metastatic tumor miceRecent studies have shown that neutrophils are the main cells in the early stage of tumor metastasis and have the role of promoting tumor metastasis.We also showed that IL-37d has the effect of inhibiting neutrophil infiltration in the inflammation model.Therefore,in order to explore the mechanism of IL-37d in tumor suppression,the LLC cell lung metastasis model was used to analyze the effect of IL-37d on early lung metastasis(1W)lung by using iodic acid Schiff staining(PAS staining)and flow cytometry.he content of neutrophils.PAS staining found that the number of neutrophil infiltration in the lungs of IL-37d group was significantly less than that of WT group;flow cytometry analysis of the number of fresh lung CD11b+Ly6G+neutrophils in mice showed that IL-37d could significantly inhibit tumors The number of lung neutrophils in the early metastasis is not affected,and it has little effect on the number of lung neutrophils in the late and advanced stages of the tumor(3 W).It showed that IL-37d mainly inhibited the recruitment of neutrophils in the lungs in the early stage of tumor metastasis(Ⅱ)The effect of IL-37d on the expression of neutrophil-related molecules in the lungs of metastatic tumor mice1.The dynamic changes of the expression of neutrophil-related molecules in the lung during the progression of lung metastasesIn order to explore the molecular mechanism of IL-37d inhibiting lung neutrophil recruitment in the early stage of tumor metastasis,we first used the tail vein injection of LLC cells to build a lung metastasis model,using real-time fluorescent quantitative PCR method,respectively 1w,2w,3w analyze the dynamic changes of these molecules.The results showed that after 1 week of tumor injection,neutrophil-related molecules conducive to tumor cell invasion-TLR3,S100a8,S100a9,MMP9 and neutrophil-related chemokines-CXCL1,CXCL2 were significantly up-regulated,while other proinflammatory cells Factors IL-6,TNF-α,and IL-1β did not increase until 3 weeks after injection of tumor cells,indicating that the lung immune microenvironment in the early stage of tumor metastasis was mainly due to changes in neutrophil-related molecules2.The effect of IL-37d on neutrophil chemokines and chemokine receptorsThe above dynamic analysis found that the neutrophil chemokines(CXCL1 and CXCL2)in the lungs at the early stage(1W)of tumor metastasis were elevated.To this end,we first tested the effect of IL-37 on the expression of CXCL1 and CXCL2,and found that in the early stage of tumor metastasis,the expression of CXCL1 and CXCL2 in the lung tissues of IL-37d transgenic mice and WT mice were higher than those of the tumor-free control mice,But there was no significant difference between the two groups,indicating that IL-37d had no significant effect on the expression of CXCL1 and CXCL2.Further,we explored the effect of IL-37d on the expression of other neutrophil chemotaxis factors(CXCL5 and CXCL12),and found that IL-37d had no significant effect on the expression of CXCL5 and CXCL12.Further,we considered that IL-37d may affect the expression of chemokine receptors and affect the sensitivity of cells to chemokines,so we also tested the effect of IL-37d on the expression of chemokine receptors-CXCR2,CXCR4 and CXCR7,it was found that IL-37d had no significant effect on the expression of these chemokine receptors in the lung.The above results indicate that IL-37d does not reduce neutrophil recruitment in the lung by inhibiting neutrophil chemokine and chemokine receptors.3.The effect of IL-37d on the expression of TLR3 in the lung during the early stage of tumor metastasisRecent studies have suggested that lung epithelial cells can sense tumor exosome RNAs via Toll-like receptor 3(TLR3)and play an important role in initiating neutrophil recruitment to the lung metastasis.Therefore,we propose a hypothesis whether IL-37d can inhibit the recruitment of neutrophils in the lung by inhibiting the expression of TLR3 in lung epithelial cells.To prove this point of view,we used real-time fluorescent quantitative PCR and immunohistochemistry to detect the expression of TLR3 in lung tissue at the early stage of metastasis(1W).The results of real-time fluorescence quantitative PCR and Western Blot showed that IL-37 had a tendency to inhibit the expression of TLR3 mRNA and protein,but did not reach statistical significance.Since TLR3 is not only expressed in lung epithelial cells,but also in dendritic cells and macrophages,in order to determine the specific cells of this change,we used immunohistochemical staining analysis,the results show that TLR3 is mainly expressed in type II In epithelial cells,the tumor-bearing mice had significantly higher TLR3 expression on lung epithelial cells compared with the control group;while IL-37d significantly inhibited the increase in TLR3 expression on mouse lung epithelial cells after tumor cell inoculation.The results showed that IL-37d mainly inhibited the expression of TLR3 on the II epithelial cells in the early stage of tumor metastasis,and thus inhibited the recruitment of neutrophils4.The effect of IL-37d on the expression of S100a8/9 and MMP9 in the lung during the early stage of tumor metastasisRecent studies have found that S100a8/9 plays an important role in the formation of tumor metastases.It not only enhances the malignancy and migration and invasion of tumor cells,but also enhances the migration ability of neutrophils and has chemotaxis.Factor-like action can recruit more neutrophils to the metastatic foci.Our dynamic analysis of the above-mentioned lung immune microenvironment found that the expression of S100a8/9 in the early stage of tumor metastasis was significantly up-regulated.In order to explore the mechanism of IL-37d inhibiting the recruitment of neutrophils in the lung in the early stage of tumor metastasis,we used real-time fluorescent quantitative PCR and immunohistochemistry to study the expression of IL-37d on these molecules at the RNA level and protein level The results of real-time fluorescence quantitative PCR(QPCR)showed that IL-37d significantly inhibited tumor-induced increase in S100a8 and S100a9 RNA transcription;Immunohisto-chemical analysis showed that IL-37d can significantly reduce the number of S100a8/9 positive cells in the lung.The above results indicate that IL-37d can inhibit the expression of S100a8 and S100a9 in the early stage of tumor metastasisS100A8 and S100A9 are known to positively regulate the expression of MMP9 and MMP9 can play an important role in the development of tumor progression and metastasis microenvironment by regulating multiple physiological processes and signaling pathways,so we used real-time fluorescent quantitative PCR,and Immunohis-tochemical methods were used to study the effect of IL-37d on MMM9 expression at RNA level and protein level.The results of real-time fluorescence quantitative PCR(QPCR)showed that IL-37d significantly inhibited the tumor-induced increase in MMP9 transcription;Immunohistochemical analysis showed that IL-37d can significantly reduce the number of MMP8 positive cells in the lung,indicating that IL-37d can also significantly inhibit the expression of MMP9 in the lung.In conclusion,IL-37d may inhibit the aggregation of neutrophils by inhibiting the expression of S100a8/9 and MMP9 in the early stage of tumor metastasis.Ⅳ.The effect and mechanism of IL-37d on tumor-induced spontaneous migration of bone marrow neutrophils(I)IL-37d can inhibit the induction of tumor cells on the spontaneous migration of bone marrow neutrophilsNew research shows that tumors can enhance the spontaneous migration of bone marrow neutrophils.This effect may be one of the mechanisms for neutrophil recruitment before metastasis.In order to further explore the mechanism of IL-37d inhibiting the recruitment of neutrophils before metastasis,we used a mouse lung cancer cell(LLC)metastasis model,by isolating and purifying Ly6G+neutrophils in bone marrow,and migrating through transwell in vitro.The experiment also used flow cytometry to analyze the number of neutrophils that had migrated to the transwell lower chamber in the absence of chemokines.The results showed that tumor cells can significantly induce the spontaneous migration ability of wild-type mouse neutrophils,IL-37d transgenic mice can significantly resist the induction of tumor cells on the spontaneous migration of bone marrow neutrophils(Ⅱ)Racl inhibitor can partially reverse the inhibitory effect of IL-37d on tumor-induced spontaneous migration of bone marrow neutrophilsThe mechanism by which tumor cells enhance the migration ability of bone marrow cells to neutrophils is still unclear.Combining the important role of Racl in promoting cell migration and the early stage of our laboratory found that IL-3 7b can inhibit Racl activity and inhibit tumor cell migration.We propose that Racl may play a role in the tumor-induced spontaneous migration of bone marrow neutrophils.We first used Racl specific inhibitor NSC23766 to block Racl activity,and conducted migration experiments.It was found that Racl inhibitors significantly inhibited the migration ability of bone marrow neutrophils.In tumor-bearing mice,the combined effect of Racl inhibitor and IL-37d is better.These preliminary results suggest that IL-37d may inhibit the spontaneous migration ability of bone marrow neutrophils by inhibiting the Racl pathway,but the exact mechanism needs further studyConclusion1.IL-37d inhibits the growth of primary subcutaneous tumors and lung metastasis of the tumor,significantly increasing the survival rate of mice2.IL-37d inhibits lung neutrophil infiltration in the early stage of tumor metastasis and inhibits the formation of metastases in the lung;the mechanism may be through the inhibition of TLR3 type Ⅱ lung epithelial cells and S100a8/9 and MMP9 expression in the lung Inhibit the accumulation of neutrophils in the lungs.3.IL-37d inhibits the spontaneous migration ability of bone marrow neutrophils,thereby inhibiting the migration of neutrophils from bone marrow to the lungs;its mechanism may play a role by inhibiting the Rac1 pathwayInnovation and significance1.We proposed for the first time that IL-37d inhibits tumor growth and metastasis by regulating the tumor microenvironment.2.First discovered and proposed that IL-37d can inhibit the migration of bone marrow neutrophils and recruitment in the lung,adjust the immune microenvironment in the lung before metastasis,and play a role in inhibiting tumor metastasis3.The results of the study revealed the formation mechanism of the immune microenvironment before tumor metastasis and the use of IL-37d to inhibit tumor metastasis and control the development of tumors,providing an experimental basis and laying a theoretical foundationLimitationDue to time constraints and the impact of the pneumonia epidemic in Xinguan,the specific mechanism of IL-37d’s ability to inhibit the spontaneous migration of bone marrow neutrophils has not been fully clarified,and in-depth research is still needed. |
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