Mechanism Of S1P Signaling Pathway Affects The Invasive Growth Of Obesity Complicated With Lymphoma

Background and purpose:Non-Hodgkin lymphoma(non-Hodgkin lymphoma,NHL)is among the top 10 malignant tumors in incidence and mortality,which is a serious threat to people’s health.With the continuous improvement of living standards,obesity has gradually become a popular health problem among people.Obesity is not only a risk factor for the development of cardiovascular disease and diabetes,but also affects the prognosis of malignant tumors including lymphoma.At present,the understanding of the impact of obesity on lymphoma is still insufficient,and various factors may affect the occurrence,development and therapeutic effect of malignant tumors in overweight patients,especially the poor therapeutic effect or drug resistance of immune checkpoint inhibitors.Obesity can result in abnormal lipid metabolism,especially abnormal sphingolipid metabolism.Sphingosine-1-phosphate(S1P)is a signaling sphingolipid.As a biologically active lipid mediator,it mediates signal transduction by binding to S1 PR receptors on the cell surface,and plays an important role in tumor cell proliferation,survival,and migration.S1 P is also a major regulator of blood vessels and the immune system,which can directly inhibit T cell immune response.Limited publications have shown that there is a relationship between abnormal S1 P metabolism and the occurrence and development of lymphoma.However,how it affects the occurrence and development of lymphomas and the interactions between tumor cells and the immune microenvironment are still unclear.This study explored to establish a mouse model of obese lymphoma to explore the mechanism of abnormal lipid metabolism on the growth and invasiveness of lymphoma;and to study how S1 P works as a cross-talk mediator between lymphoma cells and their microenvironmental immune cells and finally leads the cell growth and invasion of lymphoma;also,to explore the use of drugs to improve lipid metabolism and enhance the efficacy of immune checkpoint inhibitor PD-L1.In brief,this study provides a theoretical basis to improve the prognosis by improving lipid metabolism in the special obese lymphoma patient group.Methods:1.Human specimen experiments: immunohistochemical staining of S1 P level in human NHL microtissue array samples;for common subtypes of non-Hodgkin’s lymphoma,use GEO database to study two sphingosine kinases for S1 P biosynthesis(SPHK1 and SPHK2)gene expression.To confirm the abnormal sphingolipid metabolism in non-Hodgkin’s lymphomas.2.To establish an obese mouse model: use 6 weeks’ C57BL/6J mice,Western style high fat diet(WSHFD,35% w/w fat and 26% w/w Fructose Tong)and control diets for 16 weeks,an obese mouse model with abnormal lipid metabolism was constructed.(1)Measure the body weight and fat weight of mice,and measure the serum triglyceride level by ELISA to verify the successful modeling of the mouse model of abnormal lipid metabolism;(2)Use western blot to detect fatty acid decomposing enzymes p HSL and HSL in mouse adipose tissue;use immunohistochemistry to detect IRS and MPO in adipose tissue;explore the effects of obesity on abnormal lipid metabolism and inflammation;3.Establish a lymphoma model: Under the abnormal lipid metabolism mice,EL4 cells(T cell lymphoma)were subcutaneously planted to the mice to establish a lymphoma model.The mice were divided into two groups: EL4-CD and EL4-WSHFD.The body weight and tumor size change of mice were monitored within 21 days;the mice were sacrificed after 21 days to evaluate the tumor weight,and the extent of involvement was assessed by clinical Ann-Arbor staging according to pathology;c-MYC,CCND1,E-cadherin,Vimentin were detected by western blot in the two Group;ELISA was used to determine serum and lymphoma tissue triglyceride levels in the two groups;to explore the effect of abnormal lipid metabolism on lymphoma proliferation and EMT.4.EL4-CD and EL4-WSHFD: two groups of mice were tested by RT-PCR for lipid metabolism pathway and S1 P pathway metabolic enzymes,and Western blot and immunohistochemistry were used to test the protein expression levels of p SPHK1/SPHK1,S1PR1,S1PR3 in the two groups;discuss the influence of abnormal lipid metabolism on the exogenous uptake and endogenous synthesis of FA in lymphoma and its influence on the S1 P pathway,5.To explore the mechanism of S1 P on lymphoma proliferation and EMT: measure the expression of S1 PR in human HH,SU-DHL-4 lymphoma cell lines,blank control,S1 P,and S1 PR inhibitors to treat human HH,SU-DHL-4 lymph For tumor cell lines,proliferation was measured by XTT method,cell migration was evaluated by transwell,and YAP signal transduction pathway downstream of S1PS1 PR was measured by western blot.6.Exploring the effect of S1 P on tumor microenvironment macrophages: animal experiments: constructing a mouse model of abnormal lipid metabolism EL4 lymphoma ascites lymphoma model;detecting bone marrow-derived inhibition in the lymphoma microenvironment by immunofluorescence and flow cytometry Cells(MDSCs)CD11b+ Ly6 Chi and tumor-associated macrophages F4/80+CD206+ expression;ratio of CD4+ and CD8+ T cells.7.Exploring the mechanism of S1P-induced macrophage polarization through cell experiments: Exploring ALOX15 expression in macrophages: Exploring ALOX15 expression in EL4 mouse lymphomas with abnormal lipid metabolism through immunohistochemistry;and using human monocytes THP to stimulate differentiation into M1,M2,S1 P verified the expression of ALOX15 in M1 and M2;WT mice and Alox15-/-mice were fed with CD and WSHFD to construct a mouse model of peritoneal lymphoma.Flow cytometry was used to detect CD11b+ Ly6 Chi and peritoneal effusion.Tumor-associated macrophages F4/80+CD206+ expression;8.Establish a mouse model of EL4 lymphoma with abnormal lipid metabolism,give resveratrol and PDL1 inhibitor treatment,and explore the inhibitory effect of resveratrol on the proliferation of lymphoma and the synergistic effect of PDL1 by improving S1 P metabolism.Results:1.We performed immunohistochemical staining on paraffin sections of human NHL,and the results showed that the expression level of S1 P was higher than that of normal controls,and the expression intensity in aggressive lymphoma was higher than that in indolent lymphoma;the results of GEO database analysis showed that the expression of SPHK1 in NHL was higher The lymph nodes of the control group confirmed the presence of enhanced sphingolipid metabolism in lymphoma.2.Establish a mouse obesity model.Western Blot results show that the p HSL/HSL of adipose tissue in the WSHFD group is higher than that in the CD group;the two groups of adipose tissue immunohistochemical results indicate that the expression of insulin resistance marker IRS and inflammatory marker MPO in WSHFD group is enhanced.It suggests that there is adipose tissue decomposition and increased inflammation in obesity;3.In the mouse obese lymphoma model,the volume and weight of subcutaneous tumors in the EL4-WSHFD group were higher than those in the EL4-CD group,and there were more stages 3-4;Western Blot results showed that the EL4-WSHFD group c-MYC,CCND1,E-cadherin and Vimentin were higher than those of the control group;ELISA results showed that the triglyceride levels in serum and lymphoma tissues were higher than those in the EL4-CD group;it was confirmed that the obese group had faster lymphoma growth,stronger invasiveness,and fatty acid decomposition than the non-obese group increase.4.RT-PCR indicated that in the EL4-WSHFD group,fatty acid(FA)synthesis,FA oxidation,FA esterification and FA transport enzymes were up-regulated;the enzymes of ceramide biosynthesis S1P(ACER1,ACER3 and ASAH1)were upregulated;And up-regulation of SPHK1 for S1 P biosynthesis from sphingosine.Western Blot analysis showed that in the EL4-WSHFD group,p SPHK1/SPHK1,S1PR1,S1PR3 protein levels increased,and the SPHK1/S1P/S1PR1 and 3 signaling pathways in obese mice increased.5.Using human-derived lymphoma cells HH and SU-DHL-4 to investigate the mechanism of the above results,the results suggest that W146 blocks S1PR1 and CAY1044 blocks S1PR3,FTY720 blocks S1PR1,and SPPR3-5 significantly inhibits HH.S1P-induced cell proliferation and migration in cells and SU-DHL-4 cells.6.The immunofluorescence staining results of frozen tissues of mouse lymphoma showed that the expression of CD11b+Ly6C+ cells and F4/80+CD206+ cells in WSHFD-EL4 mice was higher than that in the CD-EL4 group.In the ascites model,the flow cytometric detection of ascites cells also obtained consistent results.Obesity affects the migration of bone marrow-derived suppressor cells to the lesion and the polarization of M2 macrophages in the tumor microenvironment.7.Immunohistochemistry confirmed that the expression of F480+ macrophages in the EL4-WSHFD group was higher than that in the EL4-CD group,and ALOX15 was co-localized with positive nuclear expression.The results of the GEO database show that the expression of ALOX15 in human specimens is higher in lymphoma than in normal lymph node tissues and is related to prognosis.In order to clarify the mechanism,S1 P treated THP-macrophages,and the results showed that the expression of ARG and TGFβ increased,and the expression of ALOX15 increased;given the S1 PR inhibitor ARG,TGFβ,and ALOX15 expression decreased.The expression of CD11b+ Ly6C+ and F4/80+CD206+ in EL4 tumor-implanted mice with Alox15 gene knockout abnormal lipid metabolism decreased.The results suggest that S1 P promotes the migration of MDSC to tumor foci,and promotes polarization to M2 by affecting the expression of TAM ALOX15.8.After EL4-WSHFD mice were treated with resveratrol,resveratrol reduced the expression of lymphoma cells SPHK1 and increased the expression of p YAP;resveratrol reduced the expression of ALOX15 and IFN.Resveratrol combined with PD-L1 inhibitors has a synergistic effect in the treatment of lymphoma.Its synergistic mechanism: resveratrol increases CD8+ T lymphocytes and CD4+IFNγ+ T lymphocytes;IFNγ inhibits the expression of ALOX15,promotes the polarization of macrophages in the tumor microenvironment to M1 and inhibits polarization to M2.Resveratrol negatively regulates the S1 P signaling pathway and inhibits the growth of lymphoma.(The above results P<0.05 are all statistically significant)Conclusions:S1P expression is increased in obese lymphoma tissue,and plays a key role in the cross-talk between lymphoma cells and their microenvironment.On the other hand,S1P/S1 PR activates the downstream YAP pathway to promote lymphoma cell proliferation and EMT.Besides,it promotes MDSC to tumors.Foci migration can promote polarization to M2 by affecting the expression of TAM ALOX15;by regulating the S1 P signaling pathway,and finally improve the prognosis of NHL.