The Immune-related Mechanism Underlying Enriched Environment-induced Tumor Growth Inhibition

Psychological stress plays important roles in the occurrence and development of tumor.Enriched environment(EE)is a kind of eustress model and previous reports have shown that EE can inhibit different tumor growth in mice.However,the mechanism of EE-induced tumor growth inhibition,especially immune-related mechanisms,is still unclear.On the basis of our previous study,the study aims to investigate the role and mechanism of NK cells and T,B lymphocytes in EE-induced tumor growth inhibition,and focus on nervous-immune axis.The present study conducts research in the following three aspects:Part One The identification of key immune cells in EE-induced tumor growth inhibitionObjective: To identify the key immue cells in EE-induced tumor growth inhibition.Methods: 1.Male,3-week-old,C57BL/6 mice,Beige mice(NK cell cytotoxicity deficiency)and Rag1-/-mice(T,B cells deficiency)were maintained under EE(EE has more space,more toys and more parters compared with SE)or SE(Standard Environment)for three weeks,then,were established Panc02 pancreatic subcutaneous xenograft model and Lewis lung cancer subcutaneous xenograft model,and tumor weight and tumor volume were observe.2.NK cells in C57BL/6 mice were depleted with anti anti-asialo GM1 antibody by intraperitoneal injection,and tumor weight and tumor volume were observed.Results: 1.In Panc02 pancreatic subcutaneous xenograft model and Lewis lung cancer subcutaneous xenograft model in wild-type C57BL/6 mice,EE could inhibit significantly tumor growth(P<0.05),and the inhibition rate was 44.3% and 39.4%,respectively.In Rag1-/-mice(T,B cells deficiency)subcutaneous xenograft model,EE also inhibited significantly tumor growth(P<0.05),and the inhibition rate was 40.4% and 36.9%,respectively.However,in Beige mice(NK cell cytotoxicity deficiency)subcutaneous xenograft model,EE has no obvious affect on tumor growth(P>0.05).2.In Rag1-/-mice(T,B cells deficiency),when NK cell were depleted with anti-asialo GM1 antibody by intraperitoneal injection,EE had no effect on Panc02 pancreatic subcutaneous xenograft growth.Conclusion: NK cell-mediated tumor immunity,not T or B cell-mediated tumor immunity plays a key role in EE-induced tumor growth inhibition.Part Two The mechanism underlying the role of NK Cells in EE-induced tumor growth inhibitionObjective: To investigate the molecule mechanism underlying the role of NK Cells in EE-induced tumor growth inhibitionMethods: Male,3-week-old,C57BL/6 mice and Rag1-/-mice were randomly placed in EE or SE for three weeks,then,were established Panc02 pancreatic subcutaneous xenograft model.The following experiments 1-3 in Wild-type C57BL/6 mice and experiments 4-5 in Rag1-/-mice were conducted: 1.The infiltration or proportion of NK cells in tumor,spleen and blood were detected by flow cytometry and immunohistochemistry,and NK cells in spleen were enriched by magnetic activated cell sorting to test NK cell cytotoxicity.2.NK cells in spleen were enriched by magnetic activated cell sorting,and chemokine receptors,killing-related receptors and effector molecules on enriched NK cells and NK cells form spleen were detected by Real-time PCR and flow cytometry,respectively.Meanwhile,expression of the corresponding ligands in tumor was tested by Real-time PCR and Western blot.3.NK cells in spleen were enriched by magnetic activated cell sorting,and NKG2 D or CCR5 on NK cells was blocked with specific antibody or blocker in vitro to observe the effect on NK cell cytotoxicity or NK cells chemotaxis enhanced by EE.4.In Panc02 pancreatic subcutaneous xenograft model of Rag1-/-mice,NKG2 D and CCR5 were blocked with specific antibody and blocker in vivo,respectively,then the influence on EE-induced tumor growth inhibition were observed,and NK cell infiltration in tumor were tested by flow cytometry when CCR5 was blocked.5.Rag1-/-mice was inoculated subcutaneously with the Panc02 cells which was lack of NKG2 D primary ligand Mult1 by RNA silencing,and EE-induced tumor inhibition were observed under the circumstances of the lack of NKG2 D ligand.Results: 1.EE could increase the proportion of NK cells in periphery(P<0.05)and promote NK infiltration in tumor(P<0.05).Meanwhile,EE could obviously enhance splenocytes and NK cell cytotoxicity to YAC-1 cells and Panc02 cells.2.EE could increase significantly the expression of activating receptor NKG2 D and chemokine receptor CCR5 on NK cells of periphery and tumor(P<0.05).However,EE has not obviously influence on expression of NKG2 D ligands and CCR5 ligands on tumor cells.3.When NKG2 D on NK cells were blocked with specific antibody in vitro,the role of EE-enhanced NK cytotoxicity to YAC-1cells and Panc02 cells was eliminated.Meanwhile,when CCR5 on NK cells were blocked with blocker DAPTA in vitro,the role of EE-enhanced NK cell chemotactic migration was eliminated.4.When NKG2 D and CCR5 were blocked with specific antibody and blocker in vivo,respectively,the role of EE-induced tumor growth inhibition was eliminated;Meanwhile,EE could not increase infiltration of NK cells into tumor when CCR5 was blocked in vivo.5.EE has no obviously influence on the growth of Panc02 pancreatic subcutaneous xenograft which was lack of NKG2 D ligand Mult1.Conclusion: EE inhibits tumor growth through increasing the expression of NKG2 D and CCR5 on NK cells,which can promote NK cells cytotoxicity to tumor cells and NK cells infiltration into tumor.Part Three The role of sympathetic nerve system in NK cell-mediated tumor growth inhibition induced by EEObjective: To study the role of sympathetic nerve system in NK cell-mediated tumor immunity enhanced by EE.Methods: 1.Male,3-weeks-old,C57BL/6 mice,which were maintained under EE or SE for three weeks,then,were established Panc02 pancreatic subcutaneous xenograft model.Mice were treated with propranolol that blocked beta adrenaline receptor(Interaction between neurotransmitters released by sympathetic nerve and immune cell was blocked).The tumor growth inhibition and NK cell cytotoxicity induced by EE were observed.2.Male,3-weeks-old,C57BL/6 mice,which were maintained under EE or SE for three weeks,then,were established Panc02 pancreatic subcutaneous xenograft model.Mice were treated with sympathectomy that blocked sympathetic nerve(98% of spleen nerve is sympathetic nerve).The tumor growth inhibition and NK cell cytotoxicity induced by EE were observed.3.Establishment of serum transfer model: Male,3-weeks-old,C57BL/6 mice were maintained under EE or SE for three weeks.Mice were inoculated subcutaneously with Panc02 pancreatic cancer cells,and serum from EE mice and SE mice was collected after Panc02 cells were inoculated for one week and was injected into other C57BL/6 mice every other day for 4 times.NK cells of spleen from serum transfer model mice were enriched to test NK cell cytotoxicity.Results: 1.When beta-receptor was blocked by propranolol,EE-induced tumor growth inhibition and EE-enhanced NK cell cytotoxicity were eliminated.These results suggest that sympathetic nerve may participate in the regulation of EE-induced NK cell cytotoxicity and tumor growth inhibition.2.When sympathetic was cut,EE-induced tumor growth inhibition and NK cell cytotoxicity were eliminated.These results further suggest that sympathetic nerve may participate in the regulation of EE-induced NK cell cytotoxicity and tumor growth inhibition.3.When mice were injected with serum from tumor-bearing mice in EE,NK cell cytotoxicity had no significantly difference(P>0.05),and the result suggests the serum components may not be the main factor of NK cells cytotoxicity enhanced by EE.Conclusion: These results suggest that sympathetic nerve pathways may play important roles in EE-induced NK cell cytotoxicity and tumor growth inhibition.